Treating and preventing microbial infections

ABSTRACT

The invention provides methods for treating or preventing microbial (eg, bacterial) infections and means for performing these methods. In particular, treatment of infections requiring rapid and durable therapy is made possible, such as for treating acute conditions such as septicemia, sepsis, SIRS or septic shock. The invention is particularly useful, for example, for treatment of microbes such as for environmental, food and beverage use. The invention relates inter alia to methods of controlling microbiologically influenced corrosion (MIC) or biofouling of a substrate or fluid in an industrial or domestic system. The invention also useful for the treatment of pathogenic bacterial infections in subjects receiving a treatment for a disease or condition, such as a transplant or a treatment for cancer, a viral infection or an autoimmune disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/029,860, filed Sep. 23, 2020, which is a continuation of Ser. No.16/700,856, filed Dec. 2, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/967,484, filed Apr. 30, 2018, the contents ofwhich are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 786212000305SEQLIST.TXT,date recorded: May 17, 2022, size: 21,079 bytes).

TECHNICAL FIELD

The invention provides methods for treating or preventing microbial (eg,bacterial) infections and means for performing these methods. Inparticular, treatment of infections requiring rapid or durable therapyis made possible, such as for treating acute conditions such assepticemia, sepsis, SIRS or septic shock. The invention is alsoparticularly useful, for example, for treatment of microbes forenvironmental, food and beverage use. The invention relates inter aliato methods of controlling microbiologically influenced corrosion (MIC)or biofouling of a substrate or fluid in an industrial or domesticsystem.

The invention also useful for the treatment of pathogenic bacterialinfections in subjects receiving a treatment for a disease or condition,such as a transplant or a treatment for cancer, a viral infection or anautoimmune disease.

BACKGROUND

Septicaemia is an acute and serious bloodstream infection. It is alsoknown as bacteraemia, or blood poisoning. Septicaemia occurs when abacterial infection elsewhere in the body, such as in the lungs or skin,enters the bloodstream. This is dangerous because the bacteria and theirtoxins can be carried through the bloodstream to a subject's entirebody. Septicaemia can quickly become life-threatening. It must berapidly treated, such as in a hospital. If it is left untreated,septicaemia can progress to sepsis.

Septicaemia and sepsis aren't the same. Sepsis is a serious complicationof septicaemia. Sepsis is when inflammation throughout the body occurs.This inflammation can cause blood clots and block oxygen from reachingvital organs, resulting in organ failure. The US National Institutes ofHealth (NIH) estimates that over 1 million Americans get severe sepsiseach year. Between 28 and 50 percent of these patients may die from thecondition. When the inflammation occurs with extremely low bloodpressure, it's called septic shock. Septic shock is fatal in many cases.

The increase in average age of the population, more people with chronicdiseases, on immunosuppressive drugs, and increase in the number ofinvasive procedures being performed has led to an increased rate ofsepsis. People over 65 years old, particularly those who have healthissues, are even more susceptible to sepsis than any other group.According to a study published in 2006, while people aged 65 years andolder make up about 12% of the American population, they make up 65% ofsepsis cases in the hospitals.

Septicaemia is caused by an infection in a part of the body. Thisinfection is typically acute. Many types of bacteria can lead tosepticaemia. The exact source of the infection often can't bedetermined. The most common infections that lead to septicaemia are:

-   -   urinary tract infections    -   lung infections, such as pneumonia    -   kidney infections    -   infections in the abdominal area

Bacteria from these infections enter the bloodstream and multiplyrapidly, causing acute infection and immediate symptoms.

People who are already in the hospital for something else, such as asurgery, are at a higher risk of developing septicaemia. Secondaryinfections can occur while in the hospital. These infections are oftenmore dangerous because the bacteria may already be resistant toantibiotics. There is a higher risk of developing septicaemia if thesubject:

-   -   has severe wounds or burns    -   is very young or very old    -   has a compromised immune system, which can occur from diseases        such as HIV or leukaemia    -   has a urinary or intravenous catheter    -   is on mechanical ventilation    -   is receiving medical treatments that weakens the immune system,        such as chemotherapy or steroid injections

The symptoms of septicaemia usually start very quickly. Even in thefirst stages of the illness, a person can look very sick. They mayfollow an injury, surgery, or another localized (eg, confined to onelocation) infection, like pneumonia. The most common initial symptomsare:

-   -   chills    -   elevated body temperature (fever)    -   very fast respiration    -   rapid heart rate

More severe symptoms will begin to emerge as the septicaemia progresseswithout proper treatment. These include the following:

-   -   confusion or inability to think clearly    -   nausea and vomiting    -   red dots that appear on the skin    -   reduced urine volume    -   inadequate blood flow (shock)

Septicaemia that has started to affect the organs or tissue function isan acute medical emergency. It must be rapidly treated at a hospital.Many people with septicaemia are admitted to a hospital's ICU fortreatment and recovery. It is recommended to never take a “wait and see”approach or try to treat the problem at home. It is crucial to get tothe hospital right away if the subject is showing signs of septicaemia.

Septicaemia has a number of serious complications. These complicationsmay be fatal if left untreated or if treatment is delayed for too long.

Septic Shock

One complication of septicaemia is a serious drop in blood pressure.This is called septic shock. Toxins released by the bacteria in thebloodstream can cause extremely low blood flow, which may result inorgan or tissue damage. Septic shock is an acute medical emergency.People with septic shock are usually cared for in a hospital's intensivecare unit (ICU). The patient may need to be put on a ventilator, orbreathing machine, if in septic shock.

Acute Respiratory Distress Syndrome (ARDS)

Another complication of septicaemia is acute respiratory distresssyndrome (ARDS). This is a life-threatening condition that preventsenough oxygen from reaching your lungs and blood. According to theNational Heart, Lung, and Blood Institute (NHLBI), ARDS is fatal inabout one-third of cases. It often results in some level of permanentlung damage. It can also damage the brain, which can lead to memoryproblems.

Sepsis

Sepsis occurs when the body has a strong immune response to theinfection. This leads to widespread inflammation throughout the body. Itis called severe sepsis if it leads to organ failure. People withchronic diseases, such as HIV or cancer, are at a higher risk of sepsis.This is because they have a weakened immune system and cannot fight offthe infection on their own. Sepsis causes millions of deaths globallyeach year and is the most common cause of death in people who have beenhospitalized. The worldwide incidence of sepsis is estimated to be 18million cases per year. In the United States sepsis affectsapproximately 3 in 1,000 people, and severe sepsis contributes to morethan 200,000 deaths per year. Sepsis occurs in 1-2% of allhospitalizations and accounts for as much as 25% of ICU bed utilization.

Early diagnosis is necessary to properly manage sepsis, as initiation ofrapid therapy is key to reducing deaths from severe sepsis. Within thefirst three hours of suspected sepsis, diagnostic studies should includewhite blood cell counts, measuring serum lactate, and obtainingappropriate cultures before starting antibiotics, so long as this doesnot delay their use by more than 45 minutes

The most common primary sources of infection resulting in sepsis are thelungs, the abdomen, and the urinary tract. Typically, 50% of all sepsiscases start as an infection in the lungs.

Speed of treatment is essential. Two sets of blood cultures (aerobic andanaerobic) should be taken without delaying the initiation ofantibiotics. Cultures from other sites such as respiratory secretions,urine, wounds, cerebrospinal fluid, and catheter insertion sites(in-situ more than 48 hours) can be taken if infections from these sitesare suspected. In severe sepsis and septic shock, broad-spectrumantibiotics (usually two, a β-lactam antibiotic with broad coverage, orbroad-spectrum carbapenem combined with fluoroquinolones, macrolides, oraminoglycosides) are conventional. However, combination of antibioticsis not recommended for the treatment of sepsis without shock and inimmunocompromised persons unless the combination is used to broaden theanti-bacterial activity. The administration of antibiotics is importantin determining the survival of the person. Some recommend they be givenwithin one hour of making the diagnosis, stating that for every hour ofdelay in the administration of antibiotics, there is an associated 6%rise in mortality.

Early goal directed therapy (EGDT) is an approach to the management ofsevere sepsis during the initial 6 hours after diagnosis. It is astep-wise approach, with the physiologic goal of optimizing cardiacpreload, afterload, and contractility. It includes giving earlyantibiotics.

Neonatal sepsis can be difficult to diagnose as newborns may beasymptomatic. If a newborn shows signs and symptoms suggestive ofsepsis, antibiotics are immediately started and are either changed totarget a specific organism identified by diagnostic testing ordiscontinued after an infectious cause for the symptoms has been ruledout.

Approximately 20-35% of people with severe sepsis and 30-70% of peoplewith septic shock die. The Surviving Sepsis Campaign (SSC) is a globalinitiative to bring together professional organizations in reducingmortality from sepsis. Antibiotics are administered within two hours ofadmission/diagnosis. For every hour a patient is denied antibiotictherapy after the onset of septic shock, the patient's chance ofsurvival is reduced by 7.9% (Survivesepsis.org 2005)

There is, therefore, a need for a rapid treatment of acute microbialinfections, such as bacterial infections associated with septicaemia,sepsis or septic shock. It would also be advantageous if the treatmentis durable for many hours. Rapid and durable treatment of microbes isalso desirable for is for controlling microbiologically influencedcorrosion (MIC) or biofouling of a substrate in industrial and domesticsystems.

Acute bacterial infections can, in certain circumstances, behealth-threatening or even life-threatening. This may be the case, forexample, in cancer patients, transplant patients or other subjects. Theneed for the treatment of the bacterial infection can become urgent andindeed an immediate focus of attention in the medical care. It would beuseful to provide methods of treating such pathogenic bacterialinfections in a way that does not adversely undermine the efficacy ofthe cancer or other separate therapy to which the patient also needs torespond.

SUMMARY OF THE INVENTION

The invention provides a solution by using the action of programmablenuclease cutting of microbe genomes; this is different from themetabolic inhibitor and other mechanisms of action used by beta-lactamsand other conventional antibiotics for treating infections. The targetedcutting provides selective microbe killing or reduction of growth orproliferation to treat or prevent infection. Moreover, the inventorshave surprisingy found a substantial killing (by several logs) can beachieved very rapidly (eg, within 15 minutes) and sustainable effectscan be achieved (eg, for more than 1 hour, and even around 3 hours aftertreatment commenced) in some embodiments. Thus, the invention providesthe following configurations.

In a First Configuration

A programmable nuclease for use in a method of treating a microbialinfection of a subject, wherein the microbial infection is caused bymicrobes of a first species or strain and the nuclease is programmableto cut a target site comprised by the genomes of microbes that haveinfected the subject, whereby microbes of the first species or strainare killed, or growth or proliferation of the microbes is reduced, thetreatment method comprising exposing the subject to the nuclease whereinthe nuclease is programmed to cut the target site, whereby genomes ofthe microbes comprised by the subject are cut and microbial infection ofthe subject is treated.

In a Second Configuration

A plurality of viruses (eg, phage or phagemids for producing phage) foruse with a programmable nuclease in a method of treating a microbialinfection of a subject, wherein the microbial infection is caused bymicrobes of a first species or strain and the nuclease is programmableto cut a target site comprised by the genomes of microbes that haveinfected the subject, whereby microbes of the first species or strainare killed, or growth or proliferation of the microbes is reduced, thetreatment method comprising exposing the subject to the nuclease andviruses wherein the nuclease is programmed to cut the target site,whereby genomes of the microbes comprised by the subject are cut andmicrobial infection of the subject is treated;

wherein each virus comprises a copy of a nucleic acid that encodes anRNA for expression of the RNA in the subject, wherein the RNA complexeswith the nuclease to program the nuclease to cut the target site inmicrobes comprised by the subject;

wherein the viruses are capable of infecting microbes comprised by thesubject to deliver thereto the nucleic acid.

In a Third Configuration

A composition comprising a plurality of nucleic acids for programming aprogrammable nuclease in a method of treating a microbial infection of asubject, wherein the microbial infection is caused by microbes of afirst species or strain and the nuclease is programmable to cut a targetsite comprised by the genomes of microbes that have infected thesubject, whereby microbes of the first species or strain are killed, orgrowth or proliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease and the nucleic acidswherein the nuclease is programmed to cut the target site, wherebygenomes of the microbes comprised by the subject are cut and microbialinfection of the subject is treated;

wherein each nucleic acid encodes an RNA for expression of the RNA inthe subject, wherein the RNA complexes with the nuclease to program thenuclease to cut the target site in microbes comprised by the subject.

In a Fourth Configuration

A CRISPR/Cas system comprising a nuclease according to the invention foruse in the method of treatment, wherein the nuclease is a Cas nuclease(eg, a Cas 3 or 9) and the system comprises one or more guide RNAs orDNA encoding one or more guide RNAs, wherein each guide RNA is capableof programming the Cas nuclease to cut a target site comprised by thegenomes of the microbes.

In a Fifth Configuration

A method of treating a microbial infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease wherein the nuclease is programmed to cut thetarget site, whereby genomes of the microbes comprised by the subjectare cut and microbial infection of the subject is treated.

In a Sixth Configuration

A method of treating a microbial infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease and a plurality of viruses wherein the nucleaseis programmed to cut the target site, whereby genomes of the microbescomprised by the subject are cut and microbial infection of the subjectis treated; wherein each virus comprises a copy of a nucleic acid thatencodes an RNA for expression of the RNA in the subject, wherein the RNAcomplexes with the nuclease to program the nuclease to cut the targetsite in microbes comprised by the subject; wherein the viruses arecapable of infecting microbes comprised by the subject to deliverthereto the nucleic acid.

In a Seventh Configuration

A method of treating a microbial infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease and a plurality of nucleic acids wherein thenuclease is programmed to cut the target site, whereby genomes of themicrobes comprised by the subject are cut and microbial infection of thesubject is treated; wherein each virus comprises a copy of a nucleicacid that encodes an RNA for expression of the RNA in the subject,wherein the RNA complexes with the nuclease to program the nuclease tocut the target site in microbes comprised by the subject; wherein eachnucleic acid encodes an RNA for expression of the RNA in the subject,wherein the RNA complexes with the nuclease to program the nuclease tocut the target site in microbes comprised by the subject.

In a Eighth Configuration

Use of a nuclease, plurality of viruses, system, guide RNA, DNA orvector of the invention, in the manufacture of a composition forcarrying out a method of treatment as defined herein, wherein thesubject is an organism other than a human or animal.

In a Ninth Configuration

Use of a nuclease, plurality of viruses, system, guide RNA, DNA orvector of the invention, in the manufacture of a composition forcarrying out an ex vivo or in vitro a method of treatment of a microbialinfection of a substrate, wherein the microbial infection is caused bymicrobes of a first species or strain and the nuclease is programmableto cut a target site comprised by the genomes of microbes that haveinfected the substrate, whereby microbes of the first species or strainare killed, or growth or proliferation of the microbes is reduced, thetreatment method comprising exposing the subject to the nuclease whereinthe nuclease is programmed to cut the target site, whereby genomes ofthe microbes comprised by the subject are cut and acute microbialinfection of the substrate is treated.

In a Tenth Configuration

Use of a programmable nuclease in the manufacture of a composition forcarrying out an ex vivo method of treatment of a microbial infection ofa substrate, wherein the microbial infection is caused by microbes of afirst species or strain and the nuclease is programmable to cut a targetsite comprised by the genomes of microbes that have infected thesubstrate, whereby microbes of the first species or strain are killed,or growth or proliferation of the microbes is reduced, the treatmentmethod comprising exposing the subject to the nuclease wherein thenuclease is programmed to cut the target site, whereby genomes of themicrobes comprised by the subject are cut and acute microbial infectionof the substrate is treated.

In any Configuration: For example, the infection is an acute infection.For example, the infection is an acute infection that is rapidlytreated. For example, the infection is treated rapidly—for example, themethod comprises reducing the infection at least 100-fold by the first30 minutes (eg, by the first 15 minutes) of the treatment. For example,the treatment is durable—for example, the reduction in infectionpersists for at least 30 minutes immediately after the first 30 minutesof the treatment. Also, optionally a reduction of the infection by atleast 100-fold or 1000-fold is maintained for at least 60 minutes (eg,at least 120 minutes) after commencement of the treatment.Exemplification is provided below which surprisingly demonstrates these,such as a rapid killing that was durable around 3 hours after treatmentcommenced. For example, the method improves survival of the subject, orimproves survival rates in humans or human patients suffering frominfection by the microbes of the first species or strain.

The invention also provides a solution to the need for effectivetreatment of pathogenic bacterial infections in subjects undergoing acancer or other, separate therapy which must also be efficacious. Thus,the invention further provides:—

In an Eleventh Configuration

A method for treating a pathogenic bacterial infection in a human oranimal subject caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga programmable nuclease that is programmed to cut the target site,wherein the subject is suffering from a further disease or conditionother than the pathogenic bacterial infection and the method comprisesadministering a therapy to the subject for treating or preventing thefurther disease or condition, wherein the nuclease treats the infectionand the therapy is efficacious in the presence of the programmednuclease to treat or prevent the disease or condition.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) that are E. coli,Pseudomonas aeruginosa or Klebsiella bacteria, the method comprisingselectively killing first bacteria comprised by the subject by cutting atarget site comprised by the genomes of the first bacteria, wherein thecutting is carried out using a Cas nuclease that is programmed by guideRNA to cut the target site, wherein the method comprises administeringan immunotherapy to the subject for treating cancer in the patient,wherein the nuclease treats the infection and the immunotherapy isefficacious in the presence of the programmed nuclease to treat thecancer;

-   -   Wherein    -   (a) The immunotherapy comprises administering to the patient an        anti-PD-1/PD-L1 axis antibody optionally selected from        pembrolizumab, nivolumab, atezolimumab, avelumab and durvalumab;        and    -   (b) The cancer is selected from melanoma; renal cell carcinoma;        bladder cancer; a solid tumour; non-small cell lung cancer        (NSCLC); forehead and neck squamous cell carcinoma (HNSCC);        Hodgkin's lymphoma; a cancer that overexpresses PD-L1 and no        mutations in EGFR or in ALK; colorectal cancer and        hepatocellular carcinoma.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga programmable nuclease that is programmed to cut the target site,wherein the subject is suffering from a cancer and the method comprisesadministering a cancer therapy to the subject for treating the cancer,wherein the nuclease treats the infection and the cancer therapy isefficacious in the presence of the programmed nuclease to treat thecancer.

In a Twelfth Configuration

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer.

In a Thirteenth Configuration

A programmable nuclease for use in the method of the invention.

In a Fourteenth Configuration

A CRISPR/Cas system comprising a nuclease according to the 13^(th)Configuration for use in the method of the 11^(th) or 12^(th)Configuration, wherein the nuclease is a Cas nuclease (eg, a Cas 3 or 9)and the system comprises one or more guide RNAs (gRNAs) or DNA encodingone or more guide RNAs, wherein each guide RNA is capable of programmingthe Cas nuclease to cut a target site comprised by the genomes of firstbacteria.

In a Fifteenth Configuration

A guide RNA or a DNA encoding a guide RNA for use in the system ormethod of treating a pathogenic bacterial infection.

In a Sixteenth Configuration

A nucleic acid vector comprising the guide RNA or DNA.

In a Seventeenth Configuration

A pharmaceutical composition comprising a first nucleic acid vector (ora plurality thereof) encoding the nuclease and a second nucleic acidvector (or a plurality thereof) encoding the guide RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show time-kill curves for Escherichia coli (EHEC) ATCC43888strain harboring the CGV system. FIG. 1A shows that CRISPR inductionkilled 99.98% of the population in 30 minutes (black line). Growth inabsence of induction is shown in dashed lines. CRISPR was induced attime-point 0 and monitored until 60 minutes. FIG. 1B shows dilutionseries (10¹-10⁶) of drop spots (5 μl) on LB agar plates of E. coliATCC43888 harboring the CGV system after 30 minutes of induction.

FIG. 2 shows CRISPR killing of target strain Escherichia coli (EHEC)ATCC43888 in Galleria mellonella larvae. G. mellonella larvae weredelivered injections of bacteria behind the final left proleg.Approximately 1 h after the injection, CRISPR inducers were administeredbehind the final right proleg. Larvae were incubated at 37° C. for 2 hand sacrificed. Control bacteria carrying an off-target single guide RNAplasmid were also injected in the control group.

FIG. 3 shows E. coli ATCC43888 count over time.

FIG. 4 shows Kaplan-Meier survival curves of Galleria mellonella larvaeinfected with Escherichia coli (EHEC) ATCC43888. CRISPR inductionsignificantly improves survival of the larvae (black line) compared tothe off-target control carrying an off-target single guide RNA plasmid(dashed line).

FIGS. 5A-5B show time-kill curves for Escherichia coli Nissle 1917harboring the CGV system targeting pks. FIG. 5A shows that CRISPRinduction killed 99.98% of the population in 15 minutes (black line).Growth in absence of induction is shown in dashed lines. CRISPR wasinduced at time-point 0 and monitored over 3 h. FIG. 5B shows dilutionseries (10¹-10⁶) of drop spots (5 μl) on LB agar plates of E. coliNissle 1917 harboring the CCV system after 15 minutes of induction.

FIGS. 6A-6B show time-kill curves for Escherichia coli Nissle 1917harboring the CCV system targeting yapH. FIG. 6A shows that CRISPRinduction killed 99.98% of the population in 15 minutes (black line).Growth in absence of induction is shown in dashed lines. CRISPR wasinduced at time-point 0 and monitored over 3 h. FIG. 6B shows dilutionseries (10¹-10⁶) of drop spots (5 μl) on LB agar plates of E. coliNissle 1917 harboring the CGV system after 15 minutes of induction.

FIG. 7 shows complete killing of transconjugant C. difficile. Thecomplete precision killing of Clostridium difficile using agRNA-encoding CRISPR array that was delivered from a probiotic carrierbacterial species by conjugative plasmids as vectors is shown. A carrierbacterium (E. coli donor strain containing the vectors was mated withClostridium difficile which was killed upon delivery of the designedarray. This harnessed the endogenous Cas3 machinery of Clostridiumdifficile. A 100% killing of Clostridium difficile cells was achievedand is shown in this figure.

FIG. 8 shows the antibiotic treatment during ICI therapy has fataloutcomes. Kaplan Meier curve for overall survival of a validation cohortfrom the Memorial Sloan Ketterin Cancer Center including n=239 advancedNSCLC patients treated with anti-PD-L1/anti-PD-1 mAb who received (ATB,n=68) or not (no ATB, n=171) antibiotics (ATB) two months before theinjection of immune checkpoint blockade. There was a medial overallsurvival of 21.9 months in the absence of antibiotic treatment, comparedto an overall survival of 9.8 months with antibiotic treatment. So, themedian overall survival in patients treated with classical antibioticsis <50% (or >12 months shorter) that of patients not receivingantibiotic treatment.

FIGS. 9A and 9B show that the gut microbiome modulates the efficacy ofanti-PD-1 inhibition in melanoma patients (from Gopalakrishnan et al,Science 2018, 359, 97-103).

DETAILED DESCRIPTION

The approach of the present invention is different from conventionalantibiotic approaches. The present invention utilizes targeted cuttingof microbial genomes using programmed nucleases, whereas conventionalantibiotics rely upon metabolic processes and cell replicationcycles—and the inhibition of these—for their activity. By focusinginstead on nuclease cutting, the invention surprisingly achieves veryquick and efficient microbial killing that also is remarkably durable.This is demonstrated in experiments below with different microbes,different nucleases and different delivery approaches. Typically,99-100% killing was surprisingly observed many times and killing of 3-4logs was very quickly achieved and with lasting duration.

The invention provides methods for treating or preventing microbial (eg,bacterial) infections and means for performing these methods. Inparticular, treatment of infections requiring rapid therapy is madepossible, such as for treating acute conditions such as septicemia,sepsis, SIRS or septic shock. As explained herein, a rapid response isvital to address microbial infection in many settings. Speed is of theessence for many infection scenarios, such as acute infections requiringhospital admission. Benefits of the invention can be one or more of: thereduction in the spread, severity or progression of the infection in thesubject; reduction in the development, severity or progression ofsymptoms of the infection (eg, sepsis or septic shock); and an increasein the likelihood of survival in human or animal patients.

The invention uses programmable nuclease cutting of microbe genomes. Thetargeted cutting provides selective microbe killing or reduction ofgrowth or proliferation to treat or prevent infection, as opposed tomore broad-spectrum microbial killing of several different species asseen with conventional antibiotics. Selective killing is advantageous toleave beneficial microbes untargeted by the treatment, which may bebeneficial to the patient. Moreover, the inventors have surprisingyfound a substantial (by several logs) killing can be achieved veryrapidly (eg, within 15 minutes) and sustainable effects can be achieved(eg, for more than 1 hour) in some embodiments. As exemplified below,the inventors surprisingly could remarkably achieve a fast and durablekilling for around 2-3 hours.

Thus, the invention provides the following aspects:—

A programmable nuclease for use in a method of treating a microbialinfection (eg, an acute bacterial infection) of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease wherein the nuclease is programmed to cut thetarget site, whereby genomes of the microbes comprised by the subjectare cut and microbial infection of the subject is treated.

Another aspect provides: A programmable nuclease for use in a method ofrapidly treating an acute microbial (eg, bacterial) infection of asubject, wherein the microbial infection is caused by microbes of afirst species or strain and the nuclease is programmable to cut a targetsite comprised by the genomes of microbes that have infected thesubject, whereby microbes of the first species or strain are killed, orgrowth or proliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease wherein the nuclease isprogrammed to cut the target site, whereby genomes of the microbescomprised by the subject are cut and acute microbial infection of thesubject is rapidly treated.

Another aspect provides: A programmable nuclease for use in a method oftreating a microbial (eg, bacterial) infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease and a nucleic acid that programs the nuclease torecognise and cut the target site, whereby genomes of the microbescomprised by the subject are cut and microbial infection of the subjectis treated.

Another aspect provides: A programmable nuclease for use in a method ofrapidly treating an acute microbial (eg, bacterial) infection of asubject, wherein the microbial infection is caused by microbes of afirst species or strain and the nuclease is programmable to cut a targetsite comprised by the genomes of microbes that have infected thesubject, whereby microbes of the first species or strain are killed, orgrowth or proliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease and a nucleic acid thatprograms the nuclease to recognise and cut the target site, wherebygenomes of the microbes comprised by the subject are cut and acutemicrobial infection of the subject is rapidly treated.

Another aspect provides: A programmable nuclease for use in a method ofdurably treating a microbial (eg, bacterial) infection of a subject,wherein the microbial infection is caused by microbes of a first speciesor strain and the nuclease is programmable to cut a target sitecomprised by the genomes of microbes that have infected the subject,whereby microbes of the first species or strain are durably killed, orgrowth or proliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease wherein the nuclease isprogrammed to cut the target site, whereby genomes of the microbescomprised by the subject are cut and microbial infection of the subjectis treated.

Another aspect provides: A programmable nuclease for use in a method ofdurably treating a microbial (eg, bacterial) infection of a subject,wherein the microbial infection is caused by microbes of a first speciesor strain and the nuclease is programmable to cut a target sitecomprised by the genomes of microbes that have infected the subject,whereby microbes of the first species or strain are durably killed, orgrowth or proliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease and a nucleic acid thatprograms the nuclease to recognise and cut the target site, wherebygenomes of the microbes comprised by the subject are cut and microbialinfection of the subject is treated.

Another aspect provides: A programmable nuclease for use in a method ofdurably treating an acute microbial (eg, bacterial) infection of asubject, wherein the microbial infection is caused by microbes of afirst species or strain and the nuclease is programmable to cut a targetsite comprised by the genomes of microbes that have infected thesubject, whereby microbes of the first species or strain are durablykilled, or growth or proliferation of the microbes is reduced, thetreatment method comprising exposing the subject to the nuclease and anucleic acid that programs the nuclease to recognise and cut the targetsite, whereby genomes of the microbes comprised by the subject are cutand acute microbial infection of the subject is treated.

Surprisingly, as exemplified below, a durable effect of several logs(eg, 3 or 4 logs) using a nuclease (as opposed to conventional means forconventional antibiotic killing) was observed around 3 hours after thefirst exposure of bacteria with a programmed nuclease. This aspect ofthe invention, therefore, makes possible dosing regimens for lessfrequent exposure to a programmed nuclesase (ie, less frequentadministration of a programmed nuclease, programmable nuclease and/ornucleic acid for programming the nuclease). For example, a Cas and gRNA(or DNA encoding a gRNA) for programming the nuclease are administeredwith a programmable nuclease (eg, a Cas 9 or Cas3) to the subject at afirst time (T1) and at a second time (T2); or gRNA (or DNA encoding agRNA) is administered on T1 and T2 for programming an endogenous Casnuclease (eg, a Cas9 or Cas3) of bacteria of said first species orstrain, wherein the programmed endogenous Cas cuts the genomes of thebacteria to kill the bacteria or to reduce growth or proliferation, thustreating the infection. Such less frequent dosing is convenient for thehealthcare practitioner and patient, as well as provides for economicaltherapy. Thus, optionally, the nuclease and/or nucleic acid isadministered to the subject on T1 and T2, wherein T2 is at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 24 hours after T1. For example,T2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 24 hours after T1.For example, T2 is 2-7 hours after T1. For example, T2 is 1 hour afterT1. For example, T2 is 2 hours after T1. For example, T2 is 3 hoursafter T1. For example, T2 is 4 hours after T1. For example, T2 is 5hours after T1.

Optionally, the nuclease (eg, programmed nuclease) and/or a nucleic acidthat programs the nuclease to recognise and cut the target site isadministered to the subject on T1 and T2, wherein T2 is at least 1 hour(eg, 1, 1.5, 2, 2.5 or 3 hours) after T1.

Another aspect provides: A Cas nuclease for use in a method of treatinga microbial (eg, bacterial) infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable with a guide RNA (gRNA) to cut a targetsite comprised by the genomes of microbes that have infected thesubject, whereby microbes of the first species or strain are killed, orgrowth or proliferation of the microbes is reduced, the treatment methodcomprising administering to the subject said a nucleic acid, wherein thenucleic acid is the gRNA or a DNA encoding the gRNA, thereby programmingthe nuclease to recognise and cut the target site of the microbescomprised by the subject, whereby genomes of the microbes are cut andmicrobial infection of the subject is treated, wherein the methodcomprises administering the nucleic acid to the subject on at a firsttime (T1) and at a second time (T2), whereby the subject is exposed toprogrammed nuclease on T1 and T2, and wherein T2 is no less than 1 hourafter T1.

Optionally, T2 is no less than 2 hours after T1; optionally, T2 is noless than 3 hours after T1; optionally, T2 is no less than 4 hours afterT1; optionally, T2 is no less than 5 hours after T1; optionally, T2 isno less than 6 hours after T1; optionally, T2 is no less than 7 hoursafter T1; optionally, T2 is no less than 8 hours after T1; optionally,T2 is no less than 9 hours after T1; optionally, T2 is no less than 10hours after T1; optionally, T2 is no less than 11 hours after T1;optionally, T2 is no less than 12 hours after T1; optionally, T2 is noless than 13 hours after T1; optionally, T2 is no less than 14 hoursafter T1; or optionally, T2 is no less than 24 hours after T1.Additionally or alternatively: Optionally, T2 is no more than 7 hoursafter T1; optionally, T2 is no more than 12 hours after T1; optionally,T2 is no more than 24 hours after T1; optionally, T2 is 2-7 hours afterT1; optionally, T2 is 24 hours after T1; optionally, T2 is 7 hours afterT1; optionally, T2 is 6 hours after T1; optionally, T2 is 5 hours afterT1; optionally, T2 is 4 hours after T1; optionally, T2 is 3 hours afterT1; optionally, T2 is 2 hours after T1; optionally, T2 is 1 hour afterT1. For example, T2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 24hours after T1. For example, T2 is 1-7 hours after T1; or T2 is 2-7hours after T1; or T2 is 3-7 hours after T1; or T2 is 4-7 hours afterT1; or T2 is 5-7 hours after T1; or T2 is 6-7 hours after T1.

Optionally, the method comprises reducing the infection at least100-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment. Optionally, the method comprises reducing the infection atleast 1000-fold by the first 30 minutes (eg, by the first 15 minutes) ofthe treatment. Optionally, the method comprises reducing the infectionat least 10000-fold by the first 30 minutes (eg, by the first 15minutes) of the treatment.

Optionally, the method comprises reducing the infection such that thereduction in infection persists for 30 minutes immediately after thefirst 30 minutes of the treatment. Optionally, the method comprisesreducing the infection such that a reduction in infection by at least100-fold persists for 30 minutes immediately after the first 30 minutesof the treatment. Optionally, the method comprises reducing theinfection such that a reduction in infection by at least 1000-foldpersists for 30 minutes immediately after the first 30 minutes of thetreatment. Optionally, the method comprises reducing the infection suchthat a reduction in infection by at least 10000-fold persists for 30minutes immediately after the first 30 minutes of the treatment.

Optionally, the method comprises reducing the infection at least100-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment; and wherein a reduction in infection by at least 100-foldpersists for 30 minutes immediately after the first 30 minutes of thetreatment. Optionally, the method comprises reducing the infection atleast 1000-fold by the first 30 minutes (eg, by the first 15 minutes) ofthe treatment; and wherein a reduction in infection by at least1000-fold persists for 30 minutes immediately after the first 30 minutesof the treatment. Optionally, the method comprises reducing theinfection at least 10000-fold by the first 30 minutes (eg, by the first15 minutes) of the treatment; and wherein a reduction in infection by atleast 10000-fold persists for 30 minutes immediately after the first 30minutes of the treatment.

Optionally, the method comprises maintaining reduction of the infectionby at least 100-fold for at least 60 minutes (eg, at least 120, 145 or180 minutes) after exposing the subject to the programmed nuclease.Optionally, a reduction of the infection by at least 100-fold ismaintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after exposing the subject to the programmed nuclease.Optionally, the method comprises maintaining reduction of the infectionby at least 1000-fold for at least 60 minutes (eg, at least 120, 145 or180 minutes) after exposing the subject to the programmed nuclease.Optionally, a reduction of the infection by at least 1000-fold ismaintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after exposing the subject to the programmed nuclease.Optionally, the method comprises maintaining reduction of the infectionby at least 10000-fold for at least 60 minutes (eg, at least 120, 145 or180 minutes) after exposing the subject to the programmed nuclease.Optionally, a reduction of the infection by at least 10000-fold ismaintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after exposing the subject to the programmed nuclease.

Optionally, the method comprises reducing the infection at least100-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment; and wherein reduction of the infection by at least 100-foldis maintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after exposing the subject to the programmed nuclease.Optionally, the method comprises reducing the infection at least1000-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment; and wherein reduction of the infection by at least 1000-foldis maintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after exposing the subject to the programmed nuclease.Optionally, the method comprises reducing the infection at least10000-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment; and wherein reduction of the infection by at least 10000-foldis maintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after exposing the subject to the programmed nuclease.

Optionally, the method comprises reducing the infection at least10000-fold by the first 15 minutes of the treatment; and whereinreduction of the infection by at least 10000-fold is maintained for atleast 45 minutes after exposing the subject to the programmed nuclease.This is exemplified below.

In an example, the infection is durably treated, wherein a reduction ofthe infection by at least 100-fold is maintained for at least 60 minutes(eg, at least 120, 145 or 180 minutes) after commencement of thetreatment. In an example, the infection is durably treated, wherein areduction of the infection by at least 1000-fold is maintained for atleast 60 minutes (eg, at least 120, 145 or 180 minutes) aftercommencement of the treatment. In an example, the infection is durablytreated, wherein a reduction of the infection by at least 10000-fold ismaintained for at least 60 minutes (eg, at least 120, 145 or 180minutes) after commencement of the treatment.

Optionally, the infection is reduced at least 100,000-fold by the first30 or 45 minutes of the treatment. Optionally, the infection is reducedat least 100,000-fold by the first 30 or 45 minutes of the treatment andthe reduction is maintained until the 60^(th) minute of the treatment.

Optionally, the infection is reduced at least 1000,000-fold by the first30 or 45 minutes of the treatment. Optionally, the infection is reducedat least 1000,000-fold by the first 30 or 45 minutes of the treatmentand the reduction is maintained until the 60^(th) minute of thetreatment.

Optionally, the infection is reduced at least 100-fold by the first 15minutes of the treatment. Optionally, the infection is reduced at least1000-fold by the first 15 minutes of the treatment. Optionally, theinfection is reduced at least 100-fold by the first 15 minutes of thetreatment and at least 1000-fold by the first 30 minutes of thetreatment.

For example, the reduction is maintained for at least 15 furtherminutes, eg, the infection is reduced at least 100-fold or at least1000-fold by the first 15 minutes of the treatment and the reduction ismaintained from the 15-30^(th) minute or 15-45^(th) minute of thetreatment or 15-60^(th) minute of the treatment.

For example, the infection is reduced at least 100-fold or at least1000-fold or at least 10000-fold by the first 15 minutes of thetreatment in the first 15 minutes and the reduction is maintained forfrom the 15-30^(th) minute or 15-45^(th) minute of the treatment.

Optionally, the method comprises reducing the infection at least100-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment.

Optionally the method comprises reducing the infection at least1000-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment.

Optionally the method comprises reducing the infection at least10000-fold by the first 30 minutes (eg, by the first 15 minutes) of thetreatment.

Optionally, the method comprises reducing the infection such that thereduction in infection persists for 30 minutes immediately after thefirst 30 minutes of the treatment, eg, the reduction may persist for atleast 60 minutes after the first 30 minutes of the treatment. If thetreatment is administered at time zero (T0), then the reduction ininfection may be present at 60 minutes counted after T0, and indeed maypersist after that 60 minutes. In FIGS. 1A, 5A and 6A, for example,reduction is seen at 60-180 minutes after T0. Optionally, the reductionin infection persists for at least 30 minutes after the first 30 minutesof the treatment.

In an example, the infection is reduced by at least 10, 20, 30, 40, 50,60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, eg, in the first15 minutes of treatment. In an example, the infection is reduced by atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99%, eg, in the first 30 minutes of treatment.

For determining killing or reduction in growth or proliferation of thetarget microbes, one can, for example, determine the difference in thenumber of microbes of the first species or strain in (i) a sample takenfrom the subject (eg, a blood, gut or leaf sample) immediately beforecommencement of the treatment and (ii) a sample (of the same type as thesample of (i), eg, a blood, gut or leaf sample respectively) taken fromthe subject at 30 minutes of the treatment. For example, if the microbesare bacteria, the samples may be assessed for the difference in colonyforming units (CFU)/ml sample, eg, when the samples have been plated onagar in respective petri dishes and incubated under identicalconditions. Another example may use microscopic counting of microbes insamples, or other routine methods know to the skilled addressee.

In an example, at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99% killing of the microbes is achieved by the first 30, 60, 90 or120 minutes (eg, by the first 30 minutes; or by the first 120 minutes)of the treatment. For example, wherein the subject is a human or animal,the killing is determined comparing the prevalence (eg, by standardcolony counting on an agar plate) of the microbes (eg, bacteria) in ablood sample taken immediately before commencement of the treatmentversus a sample taken after the first 15 or 30 minutes of the treatment.In an example, at least 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%killing of the microbes is achieved by the first 0.5, 1 or 2 hours ofthe treatment. In an example, at least 99% killing of the microbes isachieved by the first 30 minutes of the treatment. In an example, atleast 99% killing of the microbes is achieved by the first 2 hours ofthe treatment. In an example, 100% killing is achieved. These areexemplified below. In an embodiment, less than 100% of the microbes arekilled.

Worked examples of killing in bacteria are shown below. Surprisingly,using a programmed nuclease to target the bacteria of choice, specificcutting resulted in rapid killing of the target bacteria—at least 3 or 4logs of killing (ie, 1000- or 10,000 fold killing) could be observed invery short spaces of time and surprisingly these were sustained for atleast to an hour. Optionally, the infection is reduced at least1000-fold by the first 15, 30 or 45 minutes of the treatment.Optionally, the infection is reduced at least 1000-fold by the first 15,30 or 45 minutes of the treatment and the reduction is maintained untilthe 60^(th), 120^(th) or 180^(th) minute of the treatment. Optionally,the infection is reduced at least 10,000-fold by the first 15, 30 or 45minutes of the treatment. Optionally, the infection is reduced at least10,000-fold by the first 15, 30 or 45 minutes of the treatment and thereduction is maintained until the 60^(th), 120^(th) or 180^(th) minuteof the treatment. See, for example, exemplification in FIG. 5A.

In an example, 100% killing is achieved by 24 hours after commencementof the treatment.

In an example, the infection is reduced at least 1000-fold for 2 hoursor more (eg, for 2-3 hours). Optionally also the infection is reduced byat least 1000-fold by the first 15 or 13 minutes of the treatment.

In an example, the infection is reduced at least 10,000-fold for 2 hoursor more (eg, for 2-3 hours). Optionally also the infection is reduced byat least 10,000-fold by the first 15 or 13 minutes of the treatment.

In an example, the infection is reduced by at least 90, 91, 92, 93, 94,95, 96, 97, 98 or 99% for 1 hour; or for 1 hour or more; or for 2 hoursor more (eg, for 2-3 hours). Optionally, the infection is reduced by atleast 90% for 1 hour; or for 1 hour or more; or for 2 hours or more (eg,for 2-3 hours), and optionally by the first 30 minutes (eg, by the first15 minutes) of the treatment. Optionally, the infection is reduced by atleast 90% for 1 hour or more, and by the first 30 minutes (eg, by thefirst 15 minutes) of the treatment. Optionally, the infection is reducedby least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% by the first 15 or 13minutes of the treatment. Optionally, the infection is reduced by least90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% by the first 15 or 13 minutesof the treatment; and wherein the reduction is maintained for 1 hour ormore (eg, for 2 hours or more; or for 3 hours or more; or for about 2hours; or for 2 hours; or for about 3 hours; or for 3 hours).Exemplification below is provided, wherein the bacteria are E. coli.

Optionally, the subject is a human or animal and the microbes arebacteria (eg, E. coli or C. difficile), wherein blood infection of thesubject by the bacteria is reduced at least 100-fold by the first 30minutes (eg, by the first 15 minutes) of the treatment. Optionally, thesubject is a human or animal and the microbes are bacteria (eg, E. colior C. difficile), wherein blood infection of the subject by the bacteriais reduced at least 1000-fold by the first 30 minutes (eg, by the first15 minutes) of the treatment. Optionally, the subject is a human oranimal and the microbes are bacteria (eg, E. coli or C. difficile),wherein blood infection of the subject by the bacteria is reduced atleast 10,00-fold by the first 30 minutes (eg, by the first 15 minutes)of the treatment. Optionally, the E. coli are EHEC E. coli.

Optionally, the programmed nuclease (eg, a Cas9 or Cas3) is capable ofcutting a target site comprised by E. coli (EHEC) ATCC43888. Optionally,the programmed nuclease (eg, a Cas9 or Cas3) is capable of cutting atarget site comprised by E. coli Nissle.

Optionally, the blood of the subject is infected with from 10⁷ to 10¹²CFU/ml (eg, from 10⁷ to 10¹¹, from 10⁷ to 10¹⁰, from 10⁷ to 10⁹ or from10⁷ to 10⁸ CFU/ml) of the bacteria immediately before the treatment.

The worked example below shows improved survival using the method of theinvention in an in vivo model. In an example, therefore, the method ofthe invention is for improving survival of the subject by treating acutemicrobial infection of a subject. In an example, the programmed nucleaseherein is capable of carrying out the killing of bacteria of the firstspecies or strain in Galleria mellonella larvae in vivo infection model.

The nuclease may be, for example, a DNase (eg, a Cpf1, Cas9 or Cas3) ora RNase (eg, Cas13b).

In an example, the nuclease is an isolated or recombinant nuclease. Forexample, the nuclease is a synthetic or non-naturally occurringnuclease.

In an example, the nuclease is ex vivo, eg, in vitro. In an example, thenucleic acid is ex vivo. In an example, the guide RNA or DNA encodingguide RNA(s) herein is ex vivo, eg, in vitro.

Optionally, the nuclease is a Cas nuclease (eg, a Cpf1, CasX, CasY,Cas13b, Cas 3 or 9), a meganuclease, a TALEN (Transcriptionactivator-like effector nuclease) or zinc finger nuclease. In anexample, the Cas is a Streptococcus (eg, pyo genes or aureus) Cas9,Clostridium (eg, dificile), Salmonella (eg, typhimurium) or E. coliCas3. For example, the Cas is a spCas. In an example, the Cas9 is incombination with a tracrRNA or a DNA encoding a tracrRNA which isoperable with the Cas. For example, the tracrRNA is of the same speciesas the Cas, eg, a S. pyogenes tracrRNA or DNA encoding this.

In an example, the nuclease is a Cas 3 encoded by a nucleic acidcomprising SEQ ID NO: 9 or a sequence that is at least 80, 85, 90, 95,96, 97, 98 or 99% identical thereto. Optionally, also the bacteria areClostridium (eg, C. dificile) bacteria; or any Clostridium shown inTable 1. This is exemplified below.

In an example, the nuclease is a Cas 9 encoded by a nucleic acidcomprising SEQ ID NO: 10 or a sequence that is at least 80, 85, 90, 95,96, 97, 98 or 99% identical thereto. In an example, the nuclease is aCas 9 comprising SEQ ID NO: 11 or a sequence that is at least 80, 85,90, 95, 96, 97, 98 or 99% identical thereto. Optionally, also thebacteria are Clostridium (eg, C. dificile) bacteria; or any Clostridiumshown in Table 1. Optionally, also the bacteria are E. coli (eg, EHEC).This is exemplified below.

Optionally, the method comprises administering to the subject a RNA or anucleic acid (eg, DNA) that encodes an RNA for expression of the RNA inthe subject, wherein the RNA complexes with the nuclease to program thenuclease to cut the target site in microbes comprised by the subject.

Optionally, the nuclease is administered simultaneously or sequentiallywith the RNA or nucleic acid to the subject.

Optionally, subject comprises the nuclease prior to administration ofthe RNA or nucleic acid to the subject. For example, the nuclease is aCas nuclease that is an endogenous Cas nuclease of bacterial cells ofthe first species or strain that are comprised by the subject. Thus, inthis example, the RNA or nucleic acid may be administered to the subjectand introduced into the bacteria for programming endogenous Cascomprised by the bacteria, thereby forming programmed Cas nuclease thatcuts the target site in the genomes of the bacteria, whereby bacteriaare killed or growth or proliferation of bacteria is reduced, thustreating or preventing the infection.

Optionally, a plurality of viruses (eg, phage or phagemids) areadministered to the subject, wherein each virus comprises a copy (eg,one or more, eg, a plurality of copies) of the nucleic acid, wherein theviruses infect the microbes comprised by the subject to deliver theretothe nucleic acid. For example, viruses herein are phage or phagemidsthat infect (or are capable of infecting) the bacteria of the firstspecies or strain.

Optionally, the ratio of administered viruses:microbes comprised by thesubject is from 10 to 150. For example, the microbes are bacteria andthe ratio is from 10 to 100, ie, a multiplicity of infection (MOI) offrom 1 to 100 (eg, wherein the viruses are capable of replication, eg,are phage and not phagemid), eg, from 10 to 100. The ratio can bedetermined, for example, using a sample (eg, a blood or gut sample) froma human or animal subject immediately before the treatment anddetermining the number of microbes (eg, bacteria per ml of blood or gutsample). The amount of viruses to be administered can then be worked outaccording to the determination using the sample.

Optionally, the microbes are bacteria. Alternatively, the microbes arearchaea. Alternatively, the microbes are viruses. Alternatively, themicrobes are fungi. Alternatively, the microbes are algae.Alternatively, the microbes are protozoa.

In an example, the subject is a human and the infection is a nosocomialinfection. In an example, the subject is a plant, yeast, protist oramoeba.

Optionally, the subject is a human (eg, an adult, child, neonate,toddler, teenager, male or female) or animal (eg, a dog, cat, horse,cow, sheep, goat, salmon, chicken, turkey, pig, companion animal orlivestock animal).

In an example, the subject is a human or animal and: Optionally, theinfection is an infection of the lungs, abdomen or urinary tract. In anexample, the subject is suffering from a urinary tract infection, lunginfections, such as pneumonia, a kidney infection or an abdominalinfection. In an example, the subject is a surgery patient. In anexample, the subject is a burns patient. In an example, the subject hasan infected wound (eg, a bacterially infected wound). In an example, thepatient is suffering from AIDS or is infected by HIV. In an example, thesubject is suffering from a cancer, such as a blood cancer, eg,leukaemia, eg, AML or CML or CLL or a lymphoma. In an example, thesubject is a tissue or organ transplant patient, eg, a haematopoieticstem cell transplant or bone marrow transplant patient. In an example,the subject has a urinary or intravenous catheter. In an example, thesubject is on mechanical ventilation. In an example, the subject hasbeen receiving an immunosupressant. In an example, the subject issuffering from pneumonia. In an example, the subject is an intensivecare unit (ICU) patient. In an example, the subject is an Acuterespiratory distress syndrome (ARDS) patient. In an example, the subjectis suffering from meningitis, an infection in pregnancy, a rupturedgallbladder (a gallbladder rupture is a medical condition where thegallbladder leaks or bursts. Ruptures are commonly caused byinflammation of the gallbladder), abortion with septic shock (abortionwith septic shock can be an acute life-threatening illness),endometritis (endometritis is an inflammatory condition of the lining ofthe uterus, usually due to an infection), Acute Respiratory DistressSyndrome (Acute respiratory distress syndrome is a lung condition; itoccurs when fluid fills up the air sacs in the lungs) or cellulitis.

The increase in average age of the population, more people with chronicdiseases, on immunosuppressive drugs, and increase in the number ofinvasive procedures being performed has led to an increased rate ofsepsis. Optionally, the subject has undergone surgery, is on animmunosuppressant medication and/or is suffering from a chronic disease.

Optionally, the subject is a human over 60, 65, 70, 75 or 80 years ofage or is a paediatric patient. In an alternative, the subject is apaediatric patient (eg, a human baby or child) or adolescent. In anexample, the method treats or prevents neonatal sepsis in the subject.In an example the subject is an immune-compromised human or animal, eg,suffering from an acute viral infection, such as HIV infection; or thesubject is suffering from a cancer, eg, a blood cancer, such as aleukaemia; or the patient is a transplant patient, eg, that has receivedan organ, tissue or bone marrow transplant. In an example, the subjectis a human or animal that is positive for gram negative bacteriallipopolysaccharide or lipid A. In an example, the subject is a human oranimal that is positive for gram positive bacterial cell walllipoteichoic acid.

Optionally, the method treats or prevents septicaemia and/or sepsis (eg,septic shock) in the subject.

SIRS (Systemic Inflammatory Response Syndrome) criteria has been used todefine sepsis.

SIRS is the presence of two or more of the following: abnormal bodytemperature, heart rate, respiratory rate, or blood gas, and white bloodcell count. Sepsis is, for example, SIRS in response to an infectiousprocess. Severe sepsis is, for example, sepsis with sepsis-induced organdysfunction or tissue hypoperfusion (manifesting as hypotension,elevated lactate, or decreased urine output). Septic shock is, forexample, severe sepsis plus persistently low blood pressure, despite theadministration of intravenous fluids.

In an embodiment, the method prevents or delays progression of end-organdysfunction in the subject (when the subject is a human or animal).

Examples of end-organ dysfunction include the following:

-   -   Lungs: acute respiratory distress syndrome (ARDS)        (PaO₂/FiO₂<300)    -   Brain: encephalopathy symptoms including agitation, confusion,        coma; causes may include ischemia, bleeding, formation of blood        clots in small blood vessels, microabscesses, multifocal        necrotizing leukoencephalopathy    -   Liver: disruption of protein synthetic function manifests        acutely as progressive disruption of blood clotting due to an        inability to synthesize clotting factors and disruption of        metabolic functions leads to impaired bilirubin metabolism,        resulting in elevated unconjugated serum bilirubin levels    -   Kidney: low urine output or no urine output, electrolyte        abnormalities, or volume overload    -   Heart: systolic and diastolic heart failure, likely due to        chemical signals that depress myocyte function, cellular damage,        manifest as a troponin leak (although not necessarily ischemic        in nature)

More specific definitions of end-organ dysfunction exist for SIRS inpediatrics.

-   -   Cardiovascular dysfunction (after fluid resuscitation with at        least 40 ml/kg of crystalloid)    -   hypotension with blood pressure<5th percentile for age or        systolic blood pressure<2 standard deviations below normal for        age, or    -   vasopressor requirement, or    -   two of the following criteria:    -   unexplained metabolic acidosis with base deficit>5 mEq/1    -   lactic acidosis: serum lactate 2 times the upper limit of normal    -   oliguria (urine output<0.5 ml/kg/h)    -   prolonged capillary refill>5 seconds    -   core to peripheral temperature difference>3° C.    -   Respiratory dysfunction (in the absence of cyanotic heart        disease or known chronic lung disease)    -   the ratio of the arterial partial-pressure of oxygen to the        fraction of oxygen in the gases inspired (PaO₂/FiO₂)<300 (the        definition of acute lung injury), or    -   arterial partial-pressure of carbon dioxide (PaCO₂)>65 torr (20        mmHg) over baseline PaCO₂ (evidence of hypercapnic respiratory        failure), or    -   supplemental oxygen requirement of greater than FiO₂ 0.5 to        maintain oxygen saturation>92%    -   Neurologic dysfunction    -   Glasgow Coma Score (GCS)≤11, or    -   altered mental status with drop in GCS of 3 or more points in a        person with developmental delay/intellectual disability    -   Hematologic dysfunction    -   platelet count<80,000/mm³ or 50% drop from maximum in        chronically thrombocytopenic, or    -   international normalized ratio (INR)>2    -   Disseminated intravascular coagulation    -   Kidney dysfunction    -   serum creatinine>2 times the upper limit of normal for age or        2-fold increase in baseline creatinine in people with chronic        kidney disease    -   Liver dysfunction (only applicable to infants>1 month)    -   total serum bilirubin>4 mg/dl, or    -   alanine aminotransferase (ALT)>2 times the upper limit of normal

Table 2 sets out the criteria for a positive diagnosis of sepsis.

Optionally, the method reduces one or more symptoms in the patientselected from fever, low body temperature, rapid breathing, elevatedheart rate, confusion, confusion, metabolic acidosis, respiratoryalkalosis, low blood pressure, dysfunction of blood coagulation (such asblood clotting in one or more organs, or bruising) and oedema.Optionally, the method reduces septic shock. Optionally, the sepsis issevere sepsis.

Optionally, at the start of the treatment, the subject (eg, a human) hasa temperature of <36° C. or >38° C.; a heart rate of >90/min, arespiratory rate of >20 breaths/min or PaCO₂<43 kPa; and white bloodcell count of <4000/mm³ or >12,000/mm³.

Optionally, at the start of the treatment, the subject (eg, a human) haspresence of two or more of the following: abnormal body temperature,abnormal heart rate, abnormal respiratory rate, abnormal blood gas andabnormal white blood cell count.

Optionally, the subject is a plant. In an example, the subject is aprotist, eg, amoeba. Optionally in this example, the microbes areviruses (eg, large or gian viruses, eg, Mimiviruses). The nuclease, forexample, is a Cas and is programmable using a guide RNA delivered by avirophage that infects the virus microbes

In an example the microbes are yeast, eg, Candida.

Preferably, the microbes are bacteria. Optionally, the bacteria are grampositive bacteria. Optionally, the bacteria are Staphylococcus,Streptococcus, Enterococcus, Legionella, Heamophilus, Ghonnorhea,Acinetobacter, Escherichia, Klebsiella, Pseudomonas or Stenotrophomonasbacteria (eg, E. coli (eg, EHEC E. coli), C. dificile, V. cholera,Staphylococcus (eg, S. aureus or MRSA), Streptococcus pyogenes,Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa, Klebsiellapneumoniae bacteria).

Optionally, the first species is selected from the species in Table 1.

Optionally, the first species is enterohemorrhagic E. coli (EHEC), E.coli Serotype O157:H7 or Shiga-toxin producing E. coli (STEC)). In anexample, the bacteria are selected from

-   -   Shiga toxin-producing E. coli (STEC) (STEC may also be referred        to as Verocytotoxin-producing E. coli (VTEC);    -   Enterohemorrhagic E. coli (EHEC) (this pathotype is the one most        commonly heard about in the news in association with foodborne        outbreaks);    -   Enterotoxigenic E. coli (ETEC);    -   Enteropathogenic E. coli (EPEC);    -   Enteroaggregative E. coli (EAEC);    -   Enteroinvasive E. coli (EIEC); and    -   Diffusely adherent E. coli (DAEC).

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a humanpathogen responsible for outbreaks of bloody diarrhoea and haemolyticuremic syndrome (HUS) worldwide. Conventional antimicrobials trigger anSOS response in EHEC that promotes the release of the potent Shiga toxinthat is responsible for much of the morbidity and mortality associatedwith EHEC infection. Cattle are a natural reservoir of EHEC, andapproximately 75% of EHEC outbreaks are linked to the consumption ofcontaminated bovine-derived products. EHEC causes disease in humans butis asymptomatic in adult ruminants. Characteristics of E. coli serotypeO157:H7 (EHEC) infection includes abdominal cramps and bloody diarrhoea,as well as the life-threatening complication haemolytic uremic syndrome(HUS). Currently there is a need for a treatment for EHEC infections(Goldwater and Bettelheim, 2012). The use of conventional antibioticsexacerbates Shiga toxin-mediated cytotoxicity. In an epidemiology studyconducted by the Centers for Disease Control and Prevention, patientstreated with antibiotics for EHEC enteritis had a higher risk ofdeveloping HUS (Slutsker et al., 1998). Additional studies support thecontraindication of antibiotics in EHEC infection; children onantibiotic therapy for hemorrhagic colitis associated with EHEC had anincreased chance of developing HUS (Wong et al., 2000; Zimmerhackl,2000; Safdar et al., 2002; Tarr et al., 2005). Conventional antibioticspromote Shiga toxin production by enhancing the replication andexpression of stx genes that are encoded within a chromosomallyintegrated lambdoid prophage genome. The approach of the presentinvention relies on nuclease cutting. Stx induction also promotesphage-mediated lysis of the EHEC cell envelope, allowing for the releaseand dissemination of Shiga toxin into the environment (Karch et al.,1999; Matsushiro et al., 1999; Wagner et al., 2002). Thus,advantageously, the invention provides alternative means for treatingEHEC in human and animal subjects. This is exemplified below withsurprising results on the speed and duration of anti-EHEC actionproduced by nuclease action (as opposed to conventional antibioticaction).

In an example, the subject (eg, a human) is suffering from or at risk ofhaemolytic uremic syndrome (HUS), eg, the subject is suffering from anE. coli infection, such as an EHEC E. coli infection.

An aspect of the invention provides: A plurality of viruses (eg, phageor phagemids for producing phage) for use with the nuclease of theinvention in the method of treatment, wherein each virus comprises acopy of a nucleic acid described herein, wherein the viruses are capableof infecting microbes comprised by the subject to deliver thereto thenucleic acid.

A aspect of the invention provides: A plurality of viruses (eg, phage orphagemids for producing phage) for use with a programmable nuclease in amethod of treating a microbial infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease wherein the nuclease is programmed to cut thetarget site, whereby genomes of the microbes comprised by the subjectare cut and microbial infection of the subject is treated;

wherein each virus comprises a copy of a nucleic acid that encodes anRNA for expression of the RNA in the subject, wherein the RNA complexeswith the nuclease to program the nuclease to cut the target site inmicrobes comprised by the subject;

wherein the viruses are capable of infecting microbes comprised by thesubject to deliver thereto the nucleic acid.

Optionally, the method is for durable treatment, eg, as describedherein; and/or optionally, the infection is acute infection.

Optionally, the method is for rapid treatment, eg, as described herein;and/or optionally, the infection is acute infection.

Optionally, the nuclease is according to any nuclease of the inventionherein. Optionally, the nucleic acid is according to any nucleic acid ofthe invention herein.

Optionally, the nuclease is according to any nuclease of the inventionherein. Optionally, the nucleic acid is according to any nucleic acid ofthe invention herein.

In an alternative, when the microbes are viruses, the plurality ofviruses are phage that are capable of infecting host cells harbouringthe microbes, wherein the nucleic acids are introduced into the hostcells for expression therein of the RNA. The RNA complexes with thenuclease in the host cells to guide the nuclease to cut a target site ofthe microbes (ie, to cut viral RNA or DNA), thereby inactivating theviral microbes. For example, the microbes are viruses (eg, in amoeba; orin human or animal or plant cells) and the viruses of said plurality ofviruses are capable of targeting the microbes, whereby the nuclease isprogrammed to cut the microbes (eg, in the amoeba or in said cells).

An aspect of the invention provides: A composition comprising aplurality of nucleic acids for programming the nuclease of the inventionin the method of treatment, wherein each nucleic acid is a nucleic acidas defined herein.

An aspect of the invention provides: A composition comprising aplurality of nucleic acids for programming a programmable nuclease in amethod of treating a microbial infection of a subject, wherein themicrobial infection is caused by microbes of a first species or strainand the nuclease is programmable to cut a target site comprised by thegenomes of microbes that have infected the subject, whereby microbes ofthe first species or strain are killed, or growth or proliferation ofthe microbes is reduced, the treatment method comprising exposing thesubject to the nuclease and the nucleic acids wherein the nuclease isprogrammed to cut the target site, whereby genomes of the microbescomprised by the subject are cut and microbial infection of the subjectis treated; wherein each nucleic acid encodes an RNA for expression ofthe RNA in the subject, wherein the RNA complexes with the nuclease toprogram the nuclease to cut the target site in microbes comprised by thesubject.

Optionally, the method is for durable treatment, eg, as describedherein; and/or optionally, the infection is acute infection.

Optionally, the method is for rapid treatment, eg, as described herein;and/or optionally, the infection is acute infection.

Optionally, the nuclease is according to any nuclease of the inventionherein. Optionally, each nucleic acid is according to any nucleic acidof the invention herein.

Optionally, the composition is a pharmaceutical composition comprisingthe nucleic acids and a pharmaceutically acceptable diluent, carrier orexcipient. Optionally, the composition is for oral, intravenous,pulmonary, rectal, topical, buccal, ocular, intranasal, or subcutaneousadministration to a human or animal subject. Optionally, the compositionis a herbicide or pesticide or insecticide or nematodicide oraracnicide. Optionally, the composition is toxic to yeast. Optionally,the composition is toxic to giant viruses.

An aspect of the invention provides: A CRISPR/Cas system comprising anuclease according to the invention for use in the method of treatment,wherein the nuclease is a Cas nuclease (eg, a Cas 3 or 9 or any otherCas mentioned herein) and the system comprises one or more guide RNAs orDNA encoding one or more guide RNAs, wherein each guide RNA is capableof programming the Cas nuclease to cut a target site comprised by thegenomes of the microbes.

In an example, each guide RNA mentioned herein is a single guide RNA(ie, a chimaeric guide RNA). In another example, each guide RNAcomprises a crRNA that is hybridised to a tracrRNA.

In an example, a target site mentioned herein is comprised by anessential gene, virulence gene or antibiotic resistance gene of thebacteria. In an example, a target site mentioned herein is comprised bya multi-copy sequence (ie, a sequence that is present in more than one(eg, 2, 3, 4, 5, 6, 7, 8 or 9, or more) copies in each bacterialgenome). For example, the target site is comprised by a ribosomal RNAgene. In an example, a target site mentioned herein is comprised by aribosomal RNA gene (eg, a 23S ribosomal RNA gene), a yapH gene; or a pksgene; or homologue or orthologue thereof.

Optionally, each guide RNA herein is capable of hybridizing to aprotospacer sequence comprising the target site, wherein the protospacersequence is 15-45 nucleotides in length, eg, 15-25; 18-21; 20; or about20 nucleotides in length. Optionally, each guide RNA herein comprises aspacer sequence that is 15-45 nucleotides in length, eg, 15-25; 18-21;20; or about 20 nucleotides in length.

Optionally, each guide RNA herein is cognate to a 5′-NGG protospaceradjacent motif (PAM), eg, wherein the bacteria are E. coli. Optionally,each guide RNA herein is cognate to a 5′-CCA or 5′-CCT protospaceradjacent motif (PAM), eg, wherein the bacteria are C. dificile.

An aspect of the invention provides: A guide RNA or a DNA encoding aguide RNA for use in the system of the invention for use in the methodof treating an acute microbial infection in the subject, eg, septicaemiaor sepsis.

An aspect of the invention provides: A nucleic acid vector comprisingthe guide RNA or DNA.

Optionally, the vector is a phage, phagemid, viriophage, virus, plasmid(eg, conjugative plasmid) or transposon. The example below shows almostcomplete killing can be achieved using a conjugative plasmid as thevector. Thus, in an embodiment, each vector is a conjugative plasmidthat is delivered from carrier bacteria eg, probiotic carrier bacteriafor administration to the human or animal subject. In an example, thecarrier bacteria are Lactobacillus (eg, L. reuteri) or E. coli. This isexemplified below and achieved complete (100%) killing

An aspect of the invention provides: An anti-sepsis or anti-septicaemiacomposition for administration to a human or animal for treating sepsisor septicaemia, the composition comprising a plurality of vectors,wherein each vector a vector of the invention.

An aspect of the invention provides: A method of treating (eg, rapidlyand/or durably treating) an acute microbial infection of a subject,wherein the method is as defined herein.

An aspect of the invention provides: A method of treating (eg, rapidlyand/or durably treating) an acute microbial infection of a subject,wherein the microbial infection is caused by microbes of a first speciesor strain and the nuclease is programmable to cut a target sitecomprised by the genomes of microbes that have infected the subject,whereby microbes of the first species or strain are killed, or growth orproliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease wherein the nuclease isprogrammed to cut the target site, whereby genomes of the microbescomprised by the subject are cut and acute microbial infection of thesubject is treated (eg, rapidly and/or durably treated).

An aspect of the invention provides: A method of treating (eg, rapidlyand/or durably treating) an acute microbial infection of a subject,wherein the microbial infection is caused by microbes of a first speciesor strain and the nuclease is programmable to cut a target sitecomprised by the genomes of microbes that have infected the subject,whereby microbes of the first species or strain are killed, or growth orproliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease and a plurality ofviruses wherein the nuclease is programmed to cut the target site,whereby genomes of the microbes comprised by the subject are cut andacute microbial infection of the subject is treated (eg, rapidly and/ordurably treated); wherein each virus comprises a copy of a nucleic acidthat encodes an RNA for expression of the RNA in the subject, whereinthe RNA complexes with the nuclease to program the nuclease to cut thetarget site in microbes comprised by the subject; wherein the virusesare capable of infecting microbes comprised by the subject to deliverthereto the nucleic acid.

Optionally, the nuclease is according to any nuclease of the inventionherein. Optionally, the nucleic acid is according to any nucleic acid ofthe invention herein.

An aspect of the invention provides: A method of treating (eg, rapidlyand/or durably treating) an acute microbial infection of a subject,wherein the microbial infection is caused by microbes of a first speciesor strain and the nuclease is programmable to cut a target sitecomprised by the genomes of microbes that have infected the subject,whereby microbes of the first species or strain are killed, or growth orproliferation of the microbes is reduced, the treatment methodcomprising exposing the subject to the nuclease and a plurality ofnucleic acids wherein the nuclease is programmed to cut the target site,whereby genomes of the microbes comprised by the subject are cut andacute microbial infection of the subject is treated (eg, rapidly and/ordurably treated); wherein each virus comprises a copy of a nucleic acidthat encodes an RNA for expression of the RNA in the subject, whereinthe RNA complexes with the nuclease to program the nuclease to cut thetarget site in microbes comprised by the subject; wherein each nucleicacid encodes an RNA for expression of the RNA in the subject, whereinthe RNA complexes with the nuclease to program the nuclease to cut thetarget site in microbes comprised by the subject.

Optionally, the nuclease is according to any nuclease of the inventionherein. Optionally, each nucleic acid is according to any nucleic acidof the invention herein.

In an example, the invention is for medical or dental or ophthalmic use(eg, for treating or preventing an infection in an organism or limitingspread of the infection in an organism).

In an example, the invention is for cosmetic use (eg, use in a cosmeticproduct, eg, make-up), or for hygiene use (eg, use in a hygiene product,eg, soap).

In an example, the vectors and/or nuclease prior to administration tothe subject are comprised by a composition which is as any of thefollowing (host here refers to the microbes of the first species orstrain): In an example, the composition is a medical, ophthalmic, dentalor pharmaceutical composition (eg, comprised by an anti-host vaccine).In an example, the composition is an antimicrobial composition, eg, anantibiotic or antiviral, eg, a medicine, disinfectant or mouthwash. Inan example, the composition is a cosmetic composition (eg, face or bodymake-up composition). In an example, the composition is a herbicide. Inan example, the composition is a pesticide (eg, when the host is aBacillus (eg, thuringiensis) host). In an example, the composition is abeverage (eg, beer, wine or alcoholic beverage) additive. In an example,the composition is a food additive (eg, where the host is an E. coli,Salmonella, Listeria or Clostridium (eg, botulinum) host). In anexample, the composition is a water additive. In an example, thecomposition is a additive for aquatic animal environments (eg, in a fishtank). In an example, the composition is an oil or petrochemicalindustry composition or comprised in such a composition (eg, when thehost is a sulphate-reducing bacterium, eg, a Desulfovibrio host). In anexample, the composition is a oil or petrochemical additive. In anexample, the composition is a chemical additive. In an example, thecomposition is a disinfectant (eg, for sterilizing equipment for humanor animal use, eg, for surgical or medical use, or for baby feeding). Inan example, the composition is a personal hygiene composition for humanor animal use. In an example, the composition is a composition forenvironmental use, eg, for soil treatment or environmentaldecontamination (eg, from sewage, or from oil, a petrochemical or achemical, eg, when the host is a sulphate-reducing bacterium, eg, aDesulfovibrio host). In an example, the composition is a plant growthstimulator. In an example, the composition is a composition for use inoil, petrochemical, metal or mineral extraction. In an example, thecomposition is a fabric treatment or additive. In an example, thecomposition is an animal hide, leather or suede treatment or additive.In an example, the composition is a dye additive. In an example, thecomposition is a beverage (eg, beer or wine) brewing or fermentationadditive (eg, when the host is a Lactobacillus host). In an example, thecomposition is a paper additive. In an example, the composition is anink additive. In an example, the composition is a glue additive. In anexample, the composition is an anti-human or animal or plant parasiticcomposition. In an example, the composition is an air additive (eg, forair in or produced by air conditioning equipment, eg, where the host isa Legionella host). In an example, the composition is an anti-freezeadditive (eg, where the host is a Legionella host). In an example, thecomposition is an eyewash or ophthalmic composition (eg, a contact lensfluid). In an example, the composition is comprised by a dairy food (eg,the composition is in or is a milk or milk product; eg, wherein the hostis a Lactobacillus, Streptococcus, Lactococcus or Listeria host). In anexample, the composition is or is comprised by a domestic or industrialcleaning product (eg, where the host is an E. coli, Salmonella, Listeriaor Clostridium (eg, botulinum) host). In an example, the composition iscomprised by a fuel. In an example, the composition is comprised by asolvent (eg, other than water). In an example, the composition is abaking additive (eg, a food baking additive). In an example, thecomposition is a laboratory reagent (eg, for use in biotechnology orrecombinant DNA or RNA technology). In an example, the composition iscomprised by a fibre retting agent. In an example, the composition isfor use in a vitamin synthesis process. In an example, the compositionis an anti-crop or plant spoiling composition (eg, when the host is asaprotrophic bacterium). In an example, the composition is ananti-corrosion compound, eg, for preventing or reducing metal corrosion(eg, when the host is a sulphate-reducing bacterium, eg, a Desulfovibriohost, eg for use in reducing or preventing corrosion of oil extraction,treatment or containment equipment; metal extraction, treatment orcontainment equipment; or mineral extraction, treatment or containmentequipment). In an example, the composition is an agricultural or farmingcomposition or comprised in such a composition. In an example, thecomposition is a silage additive. The invention provides a CRISPR array,gRNA-encoding nucleotide sequence, vector or plurality of vectorsdescribed herein for use in any of the compositions described in thisparagraph or for use in any application described in this paragraph, eg,wherein the host cell is a bacterial or archaeal cell. The inventionprovides a method for any application described in this paragraph,wherein the method comprises combining a CRISPR array, gRNA-encodingnucleotide sequence, vector or plurality of the invention with a hostcell (eg, bacterial or archaeal cell). In an embodiment, the host cellis not present in or on a human (or human embryo) or animal.

Any aspect of the present invention is, for example, for an industrialor domestic use, or is used in a method for such use. For example, it isfor or used in agriculture, oil or petroleum industry, food or drinkindustry, clothing industry, packaging industry, electronics industry,computer industry, environmental industry, chemical industry, aerospaceindustry, automotive industry, biotechnology industry, medical industry,healthcare industry, dentistry industry, energy industry, consumerproducts industry, pharmaceutical industry, mining industry, cleaningindustry, forestry industry, fishing industry, leisure industry,recycling industry, cosmetics industry, plastics industry, pulp or paperindustry, textile industry, clothing industry, leather or suede oranimal hide industry, tobacco industry or steel industry.

Host cells herein refers to the microbes of the first species or strain.Optionally, any host cell(s) herein is/are bacterial or archaeal cells.In an example, the cell(s) is/are in stationary phase. In an example,the cell(s) is/are in exponential phase. In an example, the cell(s)is/are in lag phase. In an example, the cell(s) is/are wild-type cellsor naturally-occurring cells, eg, comprised by a naturally-occurringmicrobiome, eg, of a human, animal, plant, soil, water, sea, waterway orenvironment. In an example, the cell(s) is/are artificially geneticallymodified.

In an example, a plurality of vectors of the invention are introducedinto a plurality of said host cells, wherein the host cells arecomprised by a bacterial population, eg, ex vivo, in vivo or in vitro.In an example, the host cells are comprised by a microbiota populationcomprised by an organism or environment (eg, a waterway microbiota,water microbiota, human or animal gut microbiota, human or animal oralcavity microbiota, human or animal vaginal microbiota, human or animalskin or hair microbiota or human or animal armpit microbiota), thepopulation comprising first bacteria that are symbiotic or commensalwith the organism or environment and second bacteria comprising saidhost cells, wherein the host cells are detrimental (eg, pathogenic) tothe organism or environment. In an embodiment, the population is exvivo. In an example, the ratio of the first bacteria sub-population tothe second bacteria sub-population is increased. In an example, thefirst bacteria are Bacteroides (eg, B. fragalis and/or B.thetaiotamicron) bacteria. Optionally, the Bacteroides comprises one,two, three or more Bacteroides species selected from caccae, capillosus,cellulosilyticus, coprocola, coprophilus, coprosuis, distasonis, dorei,eggerthii, faecis, finegoldii, fluxus, fragalis, intestinalis,melaninogenicus, nordii, oleiciplenus, oralis, ovatus, pectinophilus,plebeius, stercoris, thetaiotaomicron, unifonnis, vulgatus andxylanisolvens. For example, the Bacteroides is or comprises B.thetaiotaomicron. For example, the Bacteroides is or comprises B.fragalis.

In an example, the host, first or second cells are any bacterial speciesdisclosed in US20160333348, GB1609811.3, PCT/EP2017/063593 and all USequivalent applications. The disclosures of these species (includingspecifically, Table 1 of PCT/EP2017/063593), are incorporated herein intheir entirety and for potential inclusion of one or more disclosurestherein in one or more claims herein.

In an example, the host cell(s) or bacterial population is harboured bya beverage or water (eg, a waterway or drinking water) for humanconsumption. In an example, the host cell(s) or said population iscomprised by a composition (eg, a medicament (eg, bacterial guttransplant), beverage, mouthwash or foodstuff) for administration to ahuman or non-human animal for populating and rebalancing the gut or oralmicrobiota thereof (eg, wherein said use of the medicament is to treator prevent a disease or condition in the human or animal). In anexample, the host cell(s) or said population are on a solid surface orcomprised by a biofilm (eg, a gut biofilm or a biofilm on an industrialapparatus). In an example of the invention for in vitro treating anindustrial or medical fluid, solid surface, apparatus or container (eg,for food, consumer goods, cosmetics, personal healthcare product,petroleum or oil production); or for treating a waterway, water, abeverage, a foodstuff or a cosmetic, wherein the host cell(s) arecomprised by or on the fluid, surface, apparatus, container, waterway,water, beverage, foodstuff or cosmetic.

In an example, the invention provides a container for medical ornutritional use, wherein the container comprises the vectors for use inthe method. For example, the container is a sterilised container, eg, aninhaler or connected to a syringe or IV needle.

In an example, the vectors or composition is for administration (or isadministered) to the human or non-human animal subject by mucosal, gut,oral, intranasal, intrarectal, intravaginal, ocular or buccaladministration.

Optionally, each host cell is of a strain or species found in humanmicrobiota, optionally wherein the host cells are mixed with cells of adifferent strain or species, wherein the different cells areEnterobacteriaceae or bacteria that are probiotic, commensal orsymbiotic with humans (eg, in the human gut. In an example, the hostcell is an E. coli or Salmonella cell.

The invention is optionally for altering the relative ratio ofsub-populations of first and second bacteria in a mixed population ofbacteria, eg, for altering human or animal microbiomes, such as for thealteration of the proportion of Bacteroidetes (eg, Bacteroides, eg,fragalis and/or thetaiotamicron), Firmicutes and/or gram positive ornegative bacteria in microbiota of a human.

In an example, the vectors or composition of the invention comprises anucleotide sequence for expressing in the host cell an endolysin forhost cell lysis, optionally wherein the endolysin is a phage phi11,phage Twort, phage P68, phage phiWMY or phage K endolysin (eg, MV-Lendolysin or P-27/HP endolysin).

In an example, the target site is comprised by a chromosome of eachmicrobe host cell, eg, wherein the sequence is comprised by anantibiotic resistance gene, virulence gene or essential gene of the hostcell. An example, provides the vectors of the invention in combinationwith an antibiotic agent (eg, a beta-lactam antibiotic), eg, wherein thevectors target a protospacer sequence comprised by an antibioticresistance gene comprised by host cell genome or episome (eg, a plasmidcomprised by the host cells). In an example, the episome is a plasmid,transposon, mobile genetic element or viral sequence (eg, phage orprophage sequence).

In an example, the target is a chromosomal sequence, an endogenous hostcell sequence, a wild-type host cell sequence, a non-viral chromosomalhost cell sequence, not an exogenous sequence and/or a non-phagesequence (ie, one more or all of these), eg, the sequence is a wild-typehost chromosomal cell sequence such as antibiotic resistance gene oressential gene sequence comprised by a host cell chromosome. In anexample, the sequence is a host cell plasmid sequence, eg, an antibioticresistance gene sequence.

Optionally, the nuclease is a Cas and the target site is comprised by aprotospacer sequence that is a adjacent a NGG, NAG, NGA, NGC, NGGNG,NNGRRT or NNAGAAW protospacer adjacent motif (PAM), eg, a AAAGAAA orTAAGAAA PAM (these sequences are written 5′ to 3′). In an embodiment,the PAM is immediately adjacent the 3′ end of the protospacer sequence.In an example, the Cas is a S. aureus, S. thermophilus or S. pyogenesCas. In an example, the Cas is Cpf1 and/or the PAM is TTN or CTA.Optionally, the Cas is a Type I (eg, Type I-A, I-B, I-C, I-D, I-E, orI-F) CRISPR system Cas. Optionally, the Cas is a Type II CRISPR systemCas. Optionally, the Cas is a Type IIII CRISPR system Cas. Optionally,the Cas is a Type IV CRISPR system Cas. Optionally, the Cas is a Type VCRISPR system Cas. Optionally, the Cas is a Type VI CRISPR system Cas.

Optionally, the nuclease is a Cas and each vector comprises a cognateCRISPR array that comprises multiple copies of the same spacer fortargeting the target site. Optionally, there is provide a vector orplurality of vectors of the invention, wherein the vector(s) comprises aplurality of CRISPR arrays of said gRNA-encoding sequences for host cellprotospacer sequence targeting, wherein the protospacers comprise thetarget site. Optionally, the or each vector comprises two, three or moreof copies of nucleic acid sequences encoding crRNAs (eg, gRNAs), whereinthe copies comprise the same spacer sequence for targeting a host celltarget site (eg, a site comprised by a virulence, resistance oressential gene sequence).

In an example, at least two target sequences are modified by Cas, forexample an antibiotic resistance gene and an essential gene. Multipletargeting in this way may be useful to reduce evolution of escape mutanthost cells.

In an example, the Cas is a wild-type endogenous host cell Cas nuclease.In an example, target site cutting is carried out by a dsDNA Casnuclease (eg, a Cas9, eg, a spCas9 or saCas9), whereby repair of the cutis by non-homologous end joining (NHEJ); alternatively the Cas is anexonuclease or Cas3

In an example, the array, gRNA-encoding sequence or vector is not incombination with a Cas endonuclease-encoding sequence that is naturallyfound in a cell together with repeat sequences of the array orgRNA-encoding sequence.

A tracrRNA sequence may be omitted from an array or vector of theinvention, for example for Cas systems of a Type that does not usetracrRNA, or an endogenous tracrRNA may be used with the cRNA encoded bythe vector.

In an example, the host target site is comprised by at least 5, 6, 7, 8,9, 10, 20, 30 or 40 contiguous nucleotides.

In an example, the or each vector comprises an exogenous promoterfunctional for transcription of the crRNA or gRNA in the microbes.

Optionally, each vector is a plasmid, cosmid, virus, a virion, phage,phagemid or prophage. For example, the invention provides a plurality ofbacteriophage comprising a plurality of vectors of the invention, eg,wherein the vectors are identical. In an example, the vector is a viralvector. Viral vectors have a particularly limited capacity for exogenousDNA insertion, thus virus packaging capacity needs to be considered.Room needs to be left for sequences encoding vital viral functions, suchas for expressing coat proteins and polymerase. In an example, thevector is a phage vector or an AAV or lentiviral vector. Phage vectorsare useful where the host is a bacterial cell. In an example, the vectoris a virus capable of infecting an archaea host cell.

Optionally, vector components are comprised by a transposon that iscapable of transfer into and/or between host cells. The transposon canbe a transposon as described in US20160333348, GB1609811.3 and all USequivalent applications; the disclosures of these, including thesespecific transposon disclosures, are incorporated herein in its entiretyand for potential inclusion of one or more disclosures therein in one ormore claims herein.

In an example, the or each vector is provided by a nanoparticle or inliposomes.

In an example, the or each host cell (or first and/or second bacteria)is a gram positive cell. In an example, the or each host cell is anEnterobacteriaceae, eg, Salmonella, Yersinia pestis, Klebsiella,Shigella, Proteus, Enterobacter, Serratia, or Citrobacter cells.Optionally, the or each cell is an E. coli (eg, E. coli K12) orSalmonella (eg, S enteric serovar typhimurium) cell. Optionally, the oreach host cell (or first and/or second bacteria) is a gram negativecell.

Optionally, the host (or first and/or second bacteria) is a mycoplasma,chlamydiae, spirochete or mycobacterium. Optionally, the host (or firstand/or second bacteria) is a Streptococcus (eg, pyogenes orthermophilus) host. Optionally, the host (or first and/or secondbacteria) is a Staphylococcus (eg, aureus, eg, MRSA) host. Optionally,the host (or first and/or second bacteria) is an E. coli (eg, O157:H7)host. Optionally, the host (or first and/or second bacteria) is aPseudomonas (eg, aeruginosa) host. Optionally, the host (or first and/orsecond bacteria) is a Vibro (eg, cholerae (eg, 0139) or vulnificus)host. Optionally, the host (or first and/or second bacteria) is aNeisseria (eg, gonnorrhoeae or meningitidis) host. Optionally, the host(or first and/or second bacteria) is a Bordetella (eg, pertussis) host.Optionally, the host (or first and/or second bacteria) is a Haemophilus(eg, influenzae) host. Optionally, the host (or first and/or secondbacteria) is a Shigella (eg, dysenteriae) host. Optionally, the host (orfirst and/or second bacteria) is a Brucella (eg, abortus) host.Optionally, the host (or first and/or second bacteria) is a Francisellahost. Optionally, the host (or first and/or second bacteria) is aXanthomonas host. Optionally, the host (or first and/or second bacteria)is a Agrobacterium host. Optionally, the host (or first and/or secondbacteria) is a Erwinia host. Optionally, the host (or first and/orsecond bacteria) is a Legionella (eg, pneumophila) host. Optionally, thehost (or first and/or second bacteria) is a Listeria (eg, monocytogenes)host. Optionally, the host (or first and/or second bacteria) is aCampylobacter (eg, jejuni) host. Optionally, the host (or first and/orsecond bacteria) is a Yersinia (eg, pestis) host. Optionally, the host(or first and/or second bacteria) is a Borelia (eg, burgdorferi) host.Optionally, the host (or first and/or second bacteria) is a Helicobacter(eg, pylori) host. Optionally, the host (or first and/or secondbacteria) is a Clostridium (eg, dificile or botulinum) host. Optionally,the host (or first and/or second bacteria) is a Erlichia (eg,chaffeensis) host. Optionally, the host (or first and/or secondbacteria) is a Salmonella (eg, typhi or enterica, eg, serotypetyphimurium, eg, DT 104) host. Optionally, the host (or first and/orsecond bacteria) is a Chlamydia (eg, pneumoniae) host. Optionally, thehost (or first and/or second bacteria) is a Parachlamydia host.Optionally, the host (or first and/or second bacteria) is aCorynebacterium (eg, amycolatum) host. Optionally, the host (or firstand/or second bacteria) is a Klebsiella (eg, pneumoniae) host.Optionally, the host (or first and/or second bacteria) is a Enterococcus(eg, faecalis or faecim, eg, linezolid-resistant) host. Optionally, thehost (or first and/or second bacteria) is a Acinetobacter (eg,baumannii, eg, multiple drug resistant) host.

Optionally, the invention is for reducing the growth or proliferation ofhost cell(s) in an environment (eg, soil, a composition comprising saidhost cells and yeast cells), human, animal or plant microbiome. This isuseful, for example, when the microbiome is naturally-occurring.

Optionally, the nuclease is a Cas and the target is comprised by aprotospacer sequence comprising at least 5, 6, 7, 8, 9 or 10 contiguousnucleotides immediately 3′ of a cognate PAM in the genome of the hostcell, wherein the PAM is selected from AWG, AAG, AGG, GAG and ATG.

Non-Medical, Ex vivo & In Vitro Uses Etc

In certain configurations, the inventive observation of rapid anddurable microbial killing and growth or proliferation inhibition usingnuclease cutting finds application in subjects other than humans andanimals (eg, to treat plants or yeast cultures), or for ex vivo or invitro treatment of substrates, such as industrial surfaces, fluids andapparatus. Thus, the invention further provides the following Concepts.Any other feature herein of the invention, its configurations, aspects,embodiments, options and examples above and elsewhere herein arecombinable mutatis mutandis with these Concepts (including for providingcombinations of features in the claims herein).

A Concept provides: Use of a nuclease, plurality of viruses, system,guide RNA, DNA or vector of the invention, in the manufacture of acomposition for carrying out a method of treatment as defined herein,wherein the subject is an organism other than a human or animal.

A Concept provides: Use of a nuclease, plurality of viruses, system,guide RNA, DNA or vector of the invention, in the manufacture of acomposition for carrying out an ex vivo or in vitro a method oftreatment of a microbial infection of a substrate, wherein the microbialinfection is caused by microbes of a first species or strain and thenuclease is programmable to cut a target site comprised by the genomesof microbes that have infected the substrate, whereby microbes of thefirst species or strain are killed, or growth or proliferation of themicrobes is reduced, the treatment method comprising exposing thesubject to the nuclease wherein the nuclease is programmed to cut thetarget site, whereby genomes of the microbes comprised by the subjectare cut and acute microbial infection of the substrate is treated.

Herein, treatment of an infection of a substrate may mean the treatmentof a bacterial population (eg, one or more colonies) on a surface of thesubstrate and/or incorporated in the material of the substrate. Forexample, the treatment may be the treatment to kill bacteria on thesurface of an industrial apparatus or equipment (eg, medical equipment,such as a scalpel or medical device or tubing). In another example, thesubstrate is a fluid (eg, a liquid or a gas), such as a medical fluid orpetroleum product in fluid form (eg, an oil or hydrocarbon fluid orliquid).

A Concept provides: Use of a programmable nuclease in the manufacture ofa composition for carrying out an ex vivo method of treatment of amicrobial infection of a substrate, wherein the microbial infection iscaused by microbes of a first species or strain and the nuclease isprogrammable to cut a target site comprised by the genomes of microbesthat have infected the substrate, whereby microbes of the first speciesor strain are killed, or growth or proliferation of the microbes isreduced, the treatment method comprising exposing the subject to thenuclease wherein the nuclease is programmed to cut the target site,whereby genomes of the microbes comprised by the subject are cut andacute microbial infection of the substrate is treated.

Optionally the nuclease (eg, programmed nuclease) and/or a nucleic acidthat programs the nuclease to recognise and cut the target site isadministered to the subject or substrate at a first time (T1) and at asecond time (T2) wherein T2 is at least 1 hour after T1. T1 and T2 maybe as defined herein.

Optionally, the infection is reduced at least 100-fold by the first 30minutes (eg, by the first 15 minutes) of the treatment. Optionally, theinfection is maintained by at least 100-fold for at least 60 minutes(eg, at least 120 minutes) after exposing the subject to the programmednuclease.

Optionally, the reduction in infection persists for 30 minutesimmediately after the first 30 minutes of the treatment.

Optionally, the method comprises administering to the subject orsubstrate a RNA or a nucleic acid that encodes an RNA for expression ofthe RNA in or on the subject or substrate, wherein the RNA complexeswith the nuclease to program the nuclease to cut the target site inmicrobes comprised by the subject or substrate.

Optionally, the nuclease is administered simultaneously or sequentiallywith the RNA or nucleic acid to the subject or substrate.

Optionally, the subject or substrate comprises the nuclease prior toadministration of the RNA or nucleic acid.

Optionally, a plurality of viruses (eg, phage) are administered to thesubject or substrate, wherein each virus comprises a copy of the nucleicacid, wherein the viruses infect the microbes comprised by the subjector substrate to deliver thereto the nucleic acid.

Optionally, the ratio of administered viruses:microbes is from 10 to150.

Optionally, the infection is reduced by at least 90% for 1 hour or more,optionally by the first 30 minutes (eg, by the first 15 minutes) of thetreatment.

Optionally, the infection is reduced at least 100-fold by the first 30minutes (eg, by the first 15 minutes) of the treatment; and whereinreduction of the infection by at least 100-fold is maintained for atleast 60 minutes (eg, at least 120, 145 or 180 minutes) after exposingthe subject or substrate to the programmed nuclease.

Optionally, the subject is a plant; or wherein the substrate is ametallic, plastic, concrete, stone, wood, glass or ceramic substrate.Optionally, the subject is a fluid (eg, a liquid or a gas).

Optionally, the microbes are bacteria or archaea. Optionally, thebacteria are gram positive bacteria. Optionally, the bacteria areStaphylococcus, Streptococcus, Enterococcus, Legionella, Heamophilus,Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas orStenotrophomonas bacteria (eg, E. coli (eg, EHEC E. coli), C. dificile,V. cholera, Staphylococcus (eg, S. aureus or MRSA), Streptococcuspyogenes, Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa,Klebsiella pneumoniae bacteria).

Optionally, the nuclease is a Cas nuclease (eg, a Cas 3 or 9), ameganuclease, a TALEN (Transcription activator-like effector nuclease)or zinc finger nuclease.

Reference is made to WO2016177682, which discusses aspects ofmicrobiologically influenced corrosion (MIC) or biofouling of substratesand discloses methods for controlling MIC or biofouling of a substrate.The methods, nucleases, arrays, RNAs, vectors and viruses disclosed inthat document can be employed in the present invention, for example forcarrying out the method or use of the present invention and thedisclosures of these parts and the substrates and bacteria disclosed inWO2016177682 are incorporated herein by reference for potentiallyproviding disclosure of features possible to be used in one or moreclaims herein.

Optionally, the use of the present invention is for controllingmicrobiologically influenced corrosion (MIC) or biofouling of asubstrate in an industrial or domestic system (eg, a system disclosed inWO2016177682, which disclosure is incorporated herein by reference). Inan example, the system comprises equipment (eg, for use in an industrialprocess) and the surface is a surface of said equipment. In an example,the biofouling comprises microbial biofilm and/or sludge formation,proliferation or maintenance. In an example, the microbes are sessile.In an example “controlling” comprises preventing, reducing oreliminating said MIC or biofouling, or reducing spread of said MIC orbiofouling in the system. Cell growth or proliferation or maintenanceis, for example, a characteristic of cell viability. Thus, in anexample, the method reduces microbe proliferation and/or maintenance.

Optionally, the microbes are comprised by a microbial biofilm that is incontact with said substrate. Optionally, said surface and host cells arein contact with a fluid, such as an aqueous liquid (eg, sea water, freshwater, stored water or potable water).

Fresh water is naturally occurring water on the Earth's surface in icesheets, ice caps, glaciers, icebergs, bogs, ponds, lakes, rivers andstreams, and underground as groundwater in aquifers and undergroundstreams. Fresh water is generally characterized by having lowconcentrations of dissolved salts and other total dissolved solids. Theterm specifically excludes sea water and brackish water, although itdoes include mineral-rich waters such as chalybeate springs. In anexample said fresh water is any of these fresh water types. Potablewater is water for human or animal (eg, livestock) consumption. In anexample, the fluid is selected from industrial cooling water wherein thesystem is a cooling system; sewage water wherein the system is a sewagetreatment or storage system; drinking water wherein the system is adrinking water processing, storage, transportation or delivery system;paper making water wherein the system is a paper manufacture orprocessing system; swimming pool water wherein the system is a swimmingpool or swimming pool water treatment or storage system; fireextinguisher water wherein the system is a fire extinguishing system; orindustrial process water in any pipe, tank, pit, pond or channel.

Optionally, the use is for controlling bacterial souring of a liquid ina reservoir or container), wherein the fluid comprises a population offirst host cells of a first microbial species that mediates saidbiofouling, the method comprising

-   -   (i) contacting the population with a plurality of vectors that        are capable of transforming or transducing the cells, each        vector comprising a CRISPR array whereby CRISPR arrays are        introduced into the host cells, wherein        -   (a) each CRISPR array comprises one or more sequences for            expression of a crRNA and a promoter for transcription of            the sequence(s) in a host cell; and        -   (b) each crRNA is capable of hybridising to a target            sequence of a host cell to guide Cas (eg, a Cas nuclease) in            the host cell to modify the target sequence (eg, to cut the            target sequence); the target sequence being a gene sequence            for mediating host cell viability; and            wherein the method comprises allowing expression of said            cRNAs in the presence of Cas in host cells, thereby            modifying target sequences in host cells, resulting in            reduction of host cell viability and control of said            biofouling.

In an example, the fluid is a liquid. In an example, the fluid is agaseous fluid.

Systems:

An example system is selected from the group consisting of a: —

Petrochemical recovery, processing, storage or transportation system;hydrocarbon recovery, processing, storage or transportation system;crude oil recovery, processing, storage or transportation system;natural gas recovery, processing, storage or transportation system, (eg,an oil well, oil rig, oil drilling equipment, oil pumping system, oilpipeline, gas rig, gas extraction equipment, gas pumping equipment, gaspipeline, oil tanker, gas tanker, oil storage equipment or gas storageequipment); Water processing or storage equipment; water reservoir (eg,potable water reservoir); Air or water conditioning (eg, cooling orheating) equipment, eg, a coolant tube, condenser or heat exchanger;Medical or surgical equipment; Environmental (eg, soil, waterway or air)treatment equipment; Paper manufacturing or recycling equipment; Powerplant, eg, a thermal or nuclear power plant; Fuel (eg, hydrocarbon fuel,eg, petroleum, diesel or LPG) storage equipment; Mining ormetallurgical, mineral or fuel recovery system, eg, a mine or miningequipment; Engineering system; Shipping equipment; Cargo or goodsstorage equipment (eg, a freight container); Food or beveragemanufacturing, processing or packaging equipment; Cleaning equipment(eg, laundry equipment, eg, a washing machine or dishwasher); Catering(eg, domestic or commercial catering) equipment; Farming equipment;Construction (eg, building, utilities infrastructure or roadconstruction) equipment; Aviation equipment; Aerospace equipment;Transportation equipment (eg, a motor vehicle (eg, a car, lorry or van);a railcar; an aircraft (eg, an aeroplane) or a marine or waterwayvehicle (eg, a boat or ship, submarine or hovercraft)); Packagingequipment, eg, consumer goods packaging equipment; or food or beveragepackaging equipment; Electronics (eg, a computer or mobile phone or anelectronics component thereof); or electronics manufacture or packagingequipment; Dentistry equipment; Industrial or domestic piping (eg, asub-sea pipe) or storage vessel (eg, a water tank or a fuel tank (eg,gasoline tank, eg, a gasoline tank of a vehicle)); Undergroundequipment; Building (eg, a dwelling or office or commercial premises orfactory or power station); Roadway; Bridge; Agricultural equipment;Factory system; Crude oil or natural gas exploration equipment; Officesystem; and a Household system.

In an example, the system is used in an industry or business selectedfrom the group consisting of agriculture, oil or petroleum industry,food or drink industry, clothing industry, packaging industry,electronics industry, computer industry, environmental industry,chemical industry, aerospace industry, automotive industry,biotechnology industry, medical industry, healthcare industry, dentistryindustry, energy industry, consumer products industry, pharmaceuticalindustry, mining industry, cleaning industry, forestry industry, fishingindustry, leisure industry, recycling industry, cosmetics industry,plastics industry, pulp or paper industry, textile industry, clothingindustry, leather or suede or animal hide industry, tobacco industry andsteel industry. In an example, the surface or fluid to be treated is asurface or fluid of equipment used in said selected industry. In anexample, the system is used in the crude oil industry. In an example,the system is used in the natural gas industry. In an example, thesystem is used in the petroleum industry. In an example, the system is asea container, platform or rig (eg, oil or gas platform or rig for useat sea or at sea), ship or boat. In an embodiment, such a system isanchored at sea; eg, non-temporarily anchored at sea, eg, has beenanchored at sea for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or more months (eg, contiguousmonths). In an embodiment, such a system is in the waters of a countryor state; eg, non-temporarily at sea in such waters, eg, has been inwaters of said country for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months (eg,contiguous months).

In an example, the substrate surface to be treated comprises stainlesssteel, carbon steel, copper, nickel, brass, aluminium, concrete, aplastic or wood. In an example, the substrate is a metal weld or join.In an example, the surface is a metallic (eg, steel or iron) ornon-metallic (eg, plastic, concrete, asphalt, wood, rubber or stone)surface. In an example, the metal is an alloy (eg, stainless steel,brass or a nickel-, zinc-, copper-, nickel- or aluminium-alloy). In anexample, the surface is a man-made polymer surface. In an example, thesurface is a substrate coating. In an example, the substrate is incontact with soil, fresh water or sea water.

In an example, the fluid is potable water; a waterway; brackish water;or a liquid fuel, eg, gasoline or diesel (eg, for a car or motorisedvehicle), LPG, kerosine, an alcohol (eg, ethanol, methanol or butanol),liquid hydrogen or liquid ammonia), in an example, the fuel is storedliquid fuel. In an example the fluid is an oil or non-aqueous liquid. Inan example, the fluid is a liquid comprised by a waterway or body ofwater, eg, sea water, fresh water, potable water, a river, a stream, apond, a lake, a reservoir, stored water (eg, in a water storage tank orcooling equipment), groundwater, well water, water in a rock formation,soil water or rainwater. In an example, the liquid is sea water. In anexample, the substrate is in contact with a liquid mentioned in thisparagraph. In an example, the fluid or liquid is selected from the groupconsisting of an oil, an aqueous solution, a hydraulic fracturing fluid,a fuel, carbon dioxide, a natural gas, an oil/water mixture, afuel/water mixture, water containing salts, ocean or sea water, brackishwater, sources of fresh water, lakes, rivers, stream, bogs, ponds,marshes, runoff from the thawing of snow or ice, springs, groundwater,aquifers, precipitation, any substance that is a liquid at ambienttemperature (eg, at rtp) and is hydrophobic but soluble in organicsolvents, hexanes, benzene, toluene, chloroform, diethyl ether,vegetable oils, petrochemical oils, crude oil, refined petrochemicalproducts, volatile essential oils, fossil fuels, gasoline, mixtures ofhydrocarbons, jet fuel, rocket fuel, biofuels. In an example the fluidis an oil/water mixture.

The terms “microbiologically influenced corrosion” or “MIC” as usedherein, unless otherwise specified, refer to processes in which anyelement (substrate) of a system is structurally compromised due to theaction of at least one member of a microbial population, eg, bacterialor archaeal population. The term “biofouling” as used herein, unlessotherwise specified, refers to processes in which microorganisms (suchas bacteria and/or archaea) accumulate on a substrate surface in contactwith a fluid (eg, water or an aqueous liquid, or a hydrocarbon, or apetrochemical). Also included is the undesirable accumulation andproliferation of microorganisms (such as bacteria and/or archaea) in afluid (eg, water or an aqueous liquid, or a hydrocarbon, or apetrochemical), ie, “souring” of the fluid. In an example, the bacteriaare comprised by ship or boat ballast water and the bacteria areenvironmentally undesirable. The term “substrate” as used herein refersto any type of surface on which cells can attach and a biofilm can formand grow or on which biofouling (eg slime or sludge formation) canoccur. The substrate may be an “industrial” substrate such as thesurface of equipment in an petrochemical, fuel, crude oil or gas pipingsystem, or a “non-industrial” (eg, domestic, eg, household or office)substrate such as a kitchen counter or a shower substrate or a gardensubstrate.

In an alternative, instead of a population of host bacterial cells, thepopulation is a population of archaeal cells of a first species.

Optionally, said fluid is an aqueous liquid (eg, sea water, fresh water,stored water or potable water).

In an alternative, instead the microbes are algal cells.

Optionally, the microbes are sulphate reducing bacteria (SRB) cells (eg,Desulfovibrio or Desulfotomaculum cells). In an example, the cells areselected from the group consisting of Desulfotomaculum nigrificans,Desulfacinum infenum, Thermodesulfobacterium mobile,Thermodesulforhabdus norvegicus, Archaeoglobus fulgidus,Desulfomicrobium apsheronum, Desulfovibrio gabonensis, Desulfovibriolongus, Desulfovibrio vietnamensis, Desulfobacterium cetonicum,Desulphomaculum halophilum, Desulfobacter vibrioformis andDesulfotomaculum thermocisternum cells. In an example, the populationcomprises a mixture of two or more of these cell species.

Optionally, the surface or fluid is comprised by a crude oil, gas orpetrochemicals recovery, processing, storage or transportationequipment. Crude oil is one of the most important energetic resources inthe world. It is used as raw material in numerous industries, includingthe refinery-petrochemical industry, where crude oil is refined throughvarious technological processes into consumer products such as gasoline,oils, paraffin oils, lubricants, asphalt, domestic fuel oil, vaseline,and polymers. Oil-derived products are also commonly used in many otherchemical processes. In an alternative, the fluid is a said consumerproduct or the surface is in contact with such a consumer product.

Optionally, the surface is in contact with sea water, a fracking liquidor liquid in a well; or wherein the fluid is sea water, a frackingliquid or liquid in a well.

Optionally, step (i) of the method comprises providing a population ofmicrobial cells of a second species (second host cells), the secondcells comprising said vectors, wherein the vectors are capable oftransfer from the second host cells to the first host cells; andcombining the second host cells with the first host cells, wherebyvectors are introduced into the first host cells. In an example, thesecond cell(s) are environmentally-, industrially-, ordomestically-acceptable in an environment (eg, in a water or soilenvironment) and the first host cell(s) are not acceptable in theenvironment.

Optionally, the first host cells are comprised by a mixture of microbialcells (eg, comprised by a microbial biofilm) before contact with saidvectors, wherein the mixture comprises cells of said second species.

Optionally, said second species is a species of Bacillus ornitrate-reducing bacteria or nitrate reducing sulfide oxidizing bacteria(NRB)

Optionally, the NRB is selected from the group consisting ofCampylobacter sp., Nitrobacter sp., Nitrosomonas sp., Thiomicrospirasp., Sulfurospirillum sp., Thauera sp., Paracoccus sp., Pseudomonas sp.,Rhodobacter sp. and Desulfovibrio sp; or comprises at least 2 of saidspecies.

Optionally, the NRB is selected from the group consisting of Nitrobactervulgaris, Nitrosomonas europea, Pseudomonas stutzeri, Pseudomonasaeruginosa, Paracoccus denitrificans, Sulfurospirillum deleyianum, andRhodobacter sphaeroides.

Optionally, the method comprises contacting the host cells of said firstspecies with a biocide simultaneously or sequentially with said vectors.In an example, the vectors and biocide are provided pre-mixed in acomposition that is contacted with the host cells.

Optionally, the biocide is selected from the group consisting oftetrakis hydroxymethyl phosphonium sulfate (THPS), glutaraldehyde,chlorine monoxide, chlorine dioxide, calcium hypochlorite, potassiumhypochlorite, sodium hypochlorite, dibromonitriloproprionamide (DBNPA),methylene bis(thiocyanate) (MBT), 2-(thiocyanomethylthio) benzothiazole(TCMTB), bronopol, 2-bromo-2-nitro-1,3-propanediol (BNPD), tributyltetradecyl phosphonium chloride (TTPC), taurinamide and derivativesthereof, phenols, quaternary ammonium salts, chlorine-containing agents,quinaldinium salts, lactones, organic dyes, thiosemicarbazones,quinones, carbamates, urea, salicylamide, carbanilide, guanide,amidines, imidazolines, acetic acid, benzoic acid, sorbic acid,propionic acid, boric acid, dehydroacetic acid, sulfurous acid, vanillicacid, p-hydroxybenzoate esters, isopropanol, propylene glycol, benzylalcohol, chlorobutanol, phenylethyl alcohol, formaldehyde, iodine andsolutions thereof, povidone-iodine, hexamethylenetetramine, noxythiolin,1-(3-chloroallyl)-3,5,7-triazo-1-azoniaadamantane chloride, taurolidine,taurultam, N-(5-nitro-2-furfurylidene)-1-amino-hydantoin,5-nitro-2-furaldehyde semicarbazone, 3,4,4′-trichlorocarbanilide,3,4′,5-tribromosalicylanilide,3-trifluoromethyl-4,4′-dichlorocarbanilide, 8-hydroxyquinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid,1,4-dihydro-1-ethyl-6-fluoro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid, hydrogen peroxide, peracetic acid, sodium oxychlorosene,parachlorometaxylenol, 2,4,4′-trichloro-2′-hydroxydiphenol, thymol,chlorhexidine, benzalkonium chloride, cetylpyridinium chloride, silversulfadiazine, silver nitrate, bromine, ozone, isothiazolones,polyoxyethylene (dimethylimino) ethylene (dimethylimino) ethylenedichloride, 2-(tert-butylamino)-4-chloro-6-ethylamino-5′-triazine(terbutylazine), and combinations thereof. In an example the biocide istetrakis hydroxymethyl phosphonium sulfate (THPS). In an example, thebiocide is a quaternary ammonium compound.

Optionally, the system is used in an industry operation selected fromthe group consisting of mining; shipping; crude oil, gas orpetrochemicals recovery or processing; hydraulic fracturing; air orwater heating or cooling; potable water production, storage or delivery;transportation of hydrocarbons; and wastewater treatment.

Optionally, the surface is a surface of equipment used in said selectedindustry; or wherein the fluid is a fluid comprised by equipment used insaid selected industry.

Optionally, the surface is a surface of kitchen, bathing or gardeningequipment; or wherein the fluid is comprised by kitchen, bathing orgardening equipment. For example, the equipment is used in a domesticsetting.

Optionally, the fluid is a potable liquid contained in a container (eg,water tank or bottle) and the surface is a surface of the container incontact with the liquid.

Optionally, each vector comprises a mobile genetic element (MGE),wherein the MGE comprises an origin of transfer (oriT) and a said CRISPRarray; wherein the MGE is capable of transfer between a host cell ofsaid first species and a further microbial host cell in said industrialor domestic system. For example, the further cell(s) areenvironmentally-, industrially-, or domestically-acceptable in anenvironment (eg, in a water or soil environment) and the first hostcell(s) are not acceptable in the environment. Optionally, the oriT isfunctional in the first and further host cells.

Optionally, the first and further host cells are comprised by a biofilmof fluid in contact with said surface; or wherein said cells arecomprised by said fluid.

Optionally, each MGE is or comprises an integrative and conjugativeelement (ICE); or wherein each vector is a phage that is capable ofinfecting host cells of said first species and each MGE is a phagenucleic acid that is capable of said transfer between the cells.Optionally, each ICE is a transposon, eg, a conjugative transposon.Optionally, each vector is a plasmid, optionally comprising an MGE asdescribed herein. Optionally, the sequences are comprised by aconjugative transposon of the first cell and/or further cell.

In an example, the method is a method of controlling microbiologicallyinfluenced corrosion (MIC) or biofouling of a substrate comprised by acrude oil, gas or petrochemicals recovery, processing, storage ortransportation equipment (eg, a crude oil tanker, oil rig or oildrilling equipment), wherein a surface of the substrate is in contactwith a population of first host cells, wherein the first host cells aresulphur- or sulphate-reducing bacteria (SRB), extracellular polymericsubstance-producing bacteria (EPSB), acid-producing bacteria (APB),sulphur- or sulphide-oxidizing bacteria (SOB), iron-oxidising bacteria(IOB), manganese-oxidising bacteria (MOB), ammonia producing bacteria(AmPB) or acetate producing bacteria (AcPB) of a first species thatmediates MIC or biofouling of the substrate, wherein the surface andcell population are in contact with a liquid selected from sea water,fresh water, a fracking liquid or liquid in a well (eg, oil or naturalgas well), the method comprising

-   -   (i) contacting the cell population with vectors by mixing the        liquid with a plurality of vectors that are capable of        transforming or transducing first host cells, each vector        comprising a CRISPR array whereby CRISPR arrays are introduced        into the host cells, wherein        -   (a) each CRISPR array comprises one or more sequences for            expression of a crRNA and a promoter for transcription of            the sequence(s) in a host cell;        -   (b) each crRNA is capable of hybridising to a target            sequence of a host cell to guide Cas (eg, a Cas nuclease,            eg, a Cas9 or Cfp1) in the host cell to modify the target            sequence (eg, to cut the target sequence); the target            sequence being a gene sequence for mediating host cell            viability;        -   (c) wherein each sequence of (a) comprises a sequence            R1-S1-R1′ for expression and production of the respective            crRNA in a first host cell, wherein R1 is a first CRISPR            repeat, R1′ is a second CRISPR repeat, and R1 or RP is            optional; and S1 is a first CRISPR spacer that comprises or            consists of a nucleotide sequence that is 70, 75, 80, 85, 90            or 95% or more identical to a target sequence of a said            first host cell and    -   (ii) allowing expression of said cRNAs in the presence of Cas in        host cells, thereby modifying target sequences in host cells,        resulting in reduction of host cell viability and control of MIC        or biofouling of said substrate. In an embodiment, both R1 and        R1′ are present.

In an example, the method is a method of controlling bacterialbiofouling in ballast water of a ship or boat, wherein the watercomprises a population of first host cells of a first microbial speciesthat mediates said biofouling, the method comprising

-   -   (i) contacting the population with a plurality of vectors that        are capable of transforming or transducing the cells, each        vector comprising a CRISPR array whereby CRISPR arrays are        introduced into the host cells, wherein        -   (a) each CRISPR array comprises one or more sequences for            expression of a crRNA and a promoter for transcription of            the sequence(s) in a host cell; and        -   (b) each crRNA is capable of hybridising to a target            sequence of a host cell to guide Cas (eg, a Cas nuclease) in            the host cell to modify the target sequence (eg, to cut the            target sequence); the target sequence being a gene sequence            for mediating host cell viability; and    -   (ii) allowing expression of said cRNAs in the presence of Cas in        host cells, thereby modifying target sequences in host cells,        resulting in reduction of host cell viability and control of        said biofouling.

Optionally, the first host cells are Vibrio cholerae, E. coli orEnterococci sp cells.

Optionally, step (i) comprises mixing the ballast water with thevectors, eg, in the hull of a ship or boat. Optionally, the ship or boatis a marine vehicle and the water is sea water. Optionally, instead of aship or boat, the ballast water is comprised by a container or adrilling platform at sea, eg, an oil platform or oil rig. In an example,the ship, boat, container, platform or rig is anchored at sea (ie, nottemporarily in its location).

In an example, the method is a method of discharging ballast water froma ship or boat, wherein the discharged ballast water comprises watertreated by the method. Optionally, the water is discharged into a bodyof water, eg, a sea, ocean or waterway (eg, a river, canal, lake orreservoir) or into a container.

Paragraphs:

The invention provides the following Paragraphs, which are supported bythe Examples below:—

-   -   1. A programmable Cas (eg, Cas3 or Cas9) nuclease for use in a        method of treating E. coli or C. dificile infection of a        subject, wherein the Cas nuclease is programmable with a guide        RNA to cut a target site comprised by the genomes of E. coli        or C. dificile bacteria that have infected the subject,        whereby E. coli or C. dificile cells are killed, or growth or        proliferation of the cells is reduced, the treatment method        comprising exposing the subject to the Cas nuclease wherein the        nuclease is programmed with guide RNA to cut the target site,        whereby genomes of the E. coli or C. dificile bacteria comprised        by the subject are cut and the infection of the subject is        reduced by at least 100-fold by the first 30 minutes (eg, by the        first 15 minutes) of the treatment.    -   2. A programmable Cas (eg, Cas3 or Cas9) nuclease (optionally        according to paragraph 1) for use in a method of treating E.        coli or C. dificile infection of a subject, wherein the Cas        nuclease is programmable with a guide RNA to cut a target site        comprised by the genomes of E. coli or C. dificile bacteria that        have infected the subject, whereby E. coli or C. dificile cells        are killed, or growth or proliferation of the cells is reduced,        the treatment method comprising exposing the subject to the Cas        nuclease wherein the nuclease is programmed with guide RNA to        cut the target site, whereby genomes of the E. coli or C.        dificile bacteria comprised by the subject are cut and the        infection of the subject is reduced, wherein a reduction of the        infection by at least 100-fold is maintained for at least 60        minutes (eg, at least 120, 145 or 180 minutes) after exposing        the subject to the programmed nuclease.    -   3. The nuclease of any preceding Paragraph, wherein at least 60%        of the infection is reduced by 60 minutes after exposing the        subject to the programmed nuclease.    -   4. The nuclease of any preceding Paragraph, wherein the nuclease        (eg, programmed nuclease) and/or a nucleic acid encoding the        guide RNA is administered to the subject at a first time (T1)        and at a second time (T2) wherein T2 is at least 1 hour after        T1.    -   5. The nuclease of any preceding Paragraph, wherein the method        comprises reducing the infection such that the reduction in        infection persists for 30 minutes immediately after the first 30        minutes of the treatment.    -   6. The nuclease of any preceding Paragraph, wherein the method        comprises administering to the subject the RNA or a nucleic acid        that encodes the RNA for expression of the RNA in the subject,        wherein the RNA complexes with the nuclease to program the        nuclease to cut the target site in microbes comprised by the        subject.    -   7. The nuclease of any preceding Paragraph, wherein the nuclease        is administered simultaneously or sequentially with the RNA or        nucleic acid encoding the RNA to the subject.    -   8. The nuclease of Paragraph 7, wherein the subject comprises        the nuclease prior to administration of the RNA or nucleic acid        to the subject.    -   9. The nuclease of any preceding Paragraph, wherein a plurality        of viruses (eg, phage) are administered to the subject, wherein        each virus comprises a copy of a nucleic acid encoding the RNA,        wherein the viruses infect the microbes comprised by the subject        to deliver thereto the nucleic acid.    -   10. The nuclease of Paragraph 9, wherein the ratio of        administered viruses:microbes comprised by the subject is from        10 to 150.    -   11. The nuclease according to any preceding Paragraph, wherein        the subject is a human or animal, optionally wherein the subject        is a human over 65 years of age or is a paediatric patient.    -   12. The nuclease according to Paragraph 11, wherein the        infection is an infection of the lungs, abdomen or urinary        tract; or wherein the subject has undergone surgery, is on an        immunosuppressant medication and/or is suffering from a chronic        disease.    -   13. The nuclease according to any preceding Paragraph, wherein        the infection is reduced by at least 90% for 1 hour or more,        optionally by the first 30 minutes (eg, by the first 15 minutes)        of the treatment.    -   14. The nuclease according to any preceding Paragraph, wherein        the method comprises reducing the infection at least 100-fold by        the first 30 minutes (eg, by the first 15 minutes) of the        treatment; and wherein reduction of the infection by at least        100-fold is maintained for at least 60 minutes (eg, at least        120, 145 or 180 minutes) after exposing the subject to the        programmed nuclease.    -   15. The nuclease according to any one of Paragraphs 11 to 14,        wherein the method treats or prevents septicaemia and/or sepsis        (eg, septic shock) in the subject.    -   16. The nuclease of Paragraph 16, wherein at the start of the        treatment, the subject (eg, a human) has a temperature of        <36° C. or >38° C.; a heart rate of >90/min, a respiratory rate        of >20 breaths/min or PaCO₂<4.3 kPa; and white blood cell count        of <4000/mm³ or >12,000/mm³.    -   17. The nuclease of Paragraph 15 or 16, wherein at the start of        the treatment, the subject (eg, a human) has presence of two or        more of the following: abnormal body temperature, abnormal heart        rate, abnormal respiratory rate, abnormal blood gas and abnormal        white blood cell count.    -   18. The nuclease of any preceding Paragraph, wherein the subject        is a human or animal and the microbes are bacteria (eg, E. coli        or C. dificile), wherein blood infection of the subject by the        bacteria is reduced at least 100- or 1000-fold by the first 30        minutes (eg, by the first 15 minutes) of the treatment.    -   19. The nuclease of any one of Paragraphs 11 to 18, wherein the        blood of the subject is infected with from 10⁷ to 10¹² CFU/ml of        the bacteria immediately before the treatment.    -   20. The nuclease according to any one of Paragraphs 1 to 10,        wherein the subject is a plant.    -   21. The nuclease according to any preceding Paragraph, wherein        the bacteria are comprised by a microbiome.    -   22. The nuclease according to Paragraph 21, wherein the        microbiome comprises Lactobacillus and/or Streptococcus        bacteria.    -   23. The nuclease according to any preceding Paragraph, wherein        the E. coli are EHEC E. coli.    -   24. The nuclease according to any preceding Paragraph, wherein        the nuclease is a Cas nuclease (eg, a Cas 3 or 9), a        meganuclease, a TALEN (Transcription activator-like effector        nuclease) or zinc finger nuclease.    -   25. A plurality of viruses (eg, phage or phagemids for producing        phage) for use with the nuclease of any preceding Paragraph in        the method of treatment, wherein each virus comprises a copy of        a nucleic acid encoding the RNA, wherein the viruses are capable        of infecting microbes comprised by the subject to deliver        thereto the nucleic acid.    -   26. A composition comprising a plurality of nucleic acids for        programming the nuclease of any one of Paragraphs 1 to 24 in the        method of treatment, wherein each nucleic acid is a nucleic acid        as defined in any one of Paragraphs 6 to 9.    -   27. A CRISPR/Cas system comprising a nuclease according to any        preceding Paragraph for use in the method of treatment, wherein        the nuclease is a Cas nuclease (eg, a Cas 3 or 9) and the system        comprises one or more guide RNAs or DNA encoding one or more        guide RNAs, wherein each guide RNA is capable of programming the        Cas nuclease to cut a target site comprised by the genomes of        the microbes.    -   28. A guide RNA or a DNA encoding a guide RNA for use in the        system of Paragraph 27 for use in the method of treating an        acute microbial infection in the subject, eg, septicaemia or        sepsis.    -   29. A nucleic acid vector comprising the guide RNA or DNA        recited in Paragraph 27 or 28.    -   30. The vector of Paragraph 29 wherein the vector is a phage,        phagemid, viriophage, virus, plasmid (eg, conjugative plasmid)        or transposon.    -   31. An anti-sepsis or anti-septicaemia composition for        administration to a human or animal for treating sepsis or        septicaemia, the composition comprising a plurality of vectors,        wherein each vector is according to Paragraph 29 or 30.    -   32. A method of treating an acute microbial infection of a        subject, wherein the method is as defined by any preceding        Paragraph.    -   33. Use of a nuclease, plurality of viruses, system, guide RNA,        DNA or vector of any one of Paragraphs 1 to 25 and 27 to 30, in        the manufacture of a composition for carrying out a method of        treatment as defined by any preceding Paragraph, wherein the        subject is an organism other than a human or animal.    -   34. Use of a nuclease, plurality of viruses, system, guide RNA,        DNA or vector of any one of Paragraphs 1 to 25 and 27 to 30, in        the manufacture of a composition for carrying out an ex vivo        method of treatment of a microbial infection of a substrate,        wherein the microbial infection is caused by microbes of a first        species or strain and the nuclease is programmable to cut a        target site comprised by the genomes of microbes that have        infected the substrate, whereby microbes of the first species or        strain are killed, or growth or proliferation of the microbes is        reduced, the treatment method comprising exposing the subject to        the nuclease wherein the nuclease is programmed to cut the        target site, whereby genomes of the microbes comprised by the        subject are cut and acute microbial infection of the substrate        is treated.    -   35. Use of a programmable nuclease in the manufacture of a        composition for carrying out an ex vivo method of treatment of a        microbial infection of a substrate, wherein the microbial        infection is caused by microbes of a first species or strain and        the nuclease is programmable to cut a target site comprised by        the genomes of microbes that have infected the substrate,        whereby microbes of the first species or strain are killed, or        growth or proliferation of the microbes is reduced, the        treatment method comprising exposing the subject to the nuclease        wherein the nuclease is programmed to cut the target site,        whereby genomes of the microbes comprised by the subject are cut        and acute microbial infection of the substrate is treated.    -   36. The use of Paragraph 33, 34, 35, wherein the nuclease (eg,        programmed nuclease) and/or a nucleic acid that programs the        nuclease to recognise and cut the target site is administered to        the subject or substrate at a first time (T1) and at a second        time (T2) wherein T2 is at least 1 hour after T1.    -   37. The use of any one of Paragraphs 33 to 36, wherein the        infection is reduced at least 100-fold by the first 30 minutes        (eg, by the first 15 minutes) of the treatment.    -   38. The use of any one of Paragraphs 33 to 37, wherein the        reduction of the infection is maintained by at least 100-fold        for at least 60 minutes (eg, at least 120 minutes) after        exposing the subject to the programmed nuclease.    -   39. The use of any one of Paragraphs 33 to 38, wherein the        reduction in infection persists for 30 minutes immediately after        the first 30 minutes of the treatment.    -   40. The use of any one of Paragraphs 33 to 39, wherein the        method comprises administering to the subject or substrate a RNA        or a nucleic acid that encodes an RNA for expression of the RNA        in or on the subject or substrate, wherein the RNA complexes        with the nuclease to program the nuclease to cut the target site        in microbes comprised by the subject or substrate.    -   41. The use of Paragraph 40, wherein the nuclease is        administered simultaneously or sequentially with the RNA or        nucleic acid to the subject or substrate.    -   42. The use of Paragraph 40, wherein the subject or substrate        comprises the nuclease prior to administration of the RNA or        nucleic acid.    -   43. The use of any one of Paragraphs 40 to 42, wherein a        plurality of viruses (eg, phage) are administered to the subject        or substrate, wherein each virus comprises a copy of the nucleic        acid, wherein the viruses infect the microbes comprised by the        subject or substrate to deliver thereto the nucleic acid.    -   44. The use of Paragraph 43, wherein the ratio of administered        viruses:microbes is from 10 to 150.    -   45. The use of any one of Paragraphs 33 to 44, wherein the        infection is reduced by at least 90% for 1 hour or more,        optionally by the first 30 minutes (eg, by the first 15 minutes)        of the treatment.    -   46. The use of any one of Paragraphs 44 to 45, wherein the        infection is reduced at least 100-fold by the first 30 minutes        (eg, by the first 15 minutes) of the treatment; and wherein        reduction of the infection by at least 100-fold is maintained        for at least 60 minutes (eg, at least 120, 145 or 180 minutes)        after exposing the subject or substrate to the programmed        nuclease.    -   47. The use of any one of Paragraphs 33 to 46, wherein the        subject is a plant; or wherein the substrate is a metallic,        plastic, concrete, stone, wood, glass or ceramic substrate.    -   48. The use of any one of Paragraphs 33 to 47, wherein the        microbes are bacteria.    -   49. The use according to Paragraph 48, wherein the bacteria are        gram positive bacteria.    -   50. The use according to Paragraph 48 or 49, wherein the        bacteria are Staphylococcus, Streptococcus, Enterococcus,        Legionella, Heamophilus, Ghonnorhea, Acinetobacter, Escherichia,        Klebsiella, Pseudomonas or Stenotrophomonas bacteria (eg, E.        coli (eg, EHEC E. coli), C. dificile, V. cholera, Staphylococcus        (eg, S. aureus or MRSA), Streptococcus pyogenes, Acinetobacter        baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella        pneumoniae bacteria).    -   51. The use of any one of Paragraphs 33 to 50, wherein the        nuclease is a Cas nuclease (eg, a Cas 3 or 9), a meganuclease, a        TALEN (Transcription activator-like effector nuclease) or zinc        finger nuclease.        Treatment of Pathogenic Bacterial Infections

Infectious complications are a serious cause of morbidity and mortalityin cancer patients, especially those with underlying haematologicalmalignancies where autopsy studies demonstrate that approximately 60% ofdeaths are infection related. Although fewer data exist on infectiousmortality in patients with solid organ tumours, approximately 50% ofthese patients are estimated to have an infection as either the primaryor an associated cause of death (“Epidemiology of Infections in CancerPatients”, in “Infectious Complications in Cancer Patients”, SpringerInternational Publishing Switzerland (2014)). Bacterial infectionsdominate. These infectious complications remain a significant limitationof cancer treatment modalities.

The detrimental effects of classic antibiotic treatment withbroad-spectrum antibiotics have been demonstrated in immune checkpointinhibitor (ICI)-treated cancer patients. Routy et al investigated howthe gut microbiome influences efficacy of PD-1-based immunotherapyagainst epithelial tumours (Routy et al Science 2018, 359, 91-97). Inthis work, the authors also analyzed datasets for infections/antibioticuse in patients with advanced NSCLC (n=140), renal cell carcinoma(n=67), or urothelial carcinoma (n=42) who received antibody ICI againstPD-1/PD-L1 interaction after one or several prior therapies. Among thesepatients, they were prescribed broad-spectrum antibiotics(beta-lactam+/−inhibitors, fluoroquinolones, or macrolides) within 2months before, or 1 month after, the first administration of PD-1/PD-L1mAb. Patients generally took antibiotic orally for common indications(dental, urinary, and pulmonary infections). The detrimental effect oftreating infections in cancer patients undergoing ICI therapy withclassical, broad-spectrum antibiotics was observed. See FIG. 8 , whichshows that the antibiotic treatment during ICI therapy has fataloutcomes: there was a medial overall survival of 21.9 months in theabsence of antibiotic treatment, compared to an overall survival of 9.8months with antibiotic treatment. So, the median overall survival inpatients treated with classical antibiotics is <50% (or >12 monthsshorter) that of patients not receiving antibiotic treatment.

The work by Gopalakrishnan et al is another recent example lendingsupport to the importance of a “healthy” microbiome in immuno-oncologytherapy outcomes (Gopalakrishnan et al, Science 2018, 359, 97-103). SeeFIGS. 9A and 9B. It was observed that the gut microbiome modulates theefficacy of anti-PD-1 inhibition in melanoma patients.

Several other studies add to the expanding evidence base of the criticallink between the microbiome and immuno-oncology outcomes:

-   “Microbiota: a key orchestrator of cancer therapy”, Nat. Rev. Cancer    2017, 17, 271-285-   Matson et al, Science 2018, 359, 104-108-   L. Derosa et al Annals of Oncology 2018 (epub 30 Mar. 2018)-   M. Vétizou et al Science. 2015, 350, 1079-84-   Sivan et al Science 2015, 350, 1084-1089

Another report—claiming to be the first systematic review of infectionamong patients receiving immune checkpoint blockade for cancertherapy—investigated serious infections in melanoma patients treatedwith immune checkpoint inhibitors (against CTLA-4, PD-1, and/or PD-L1)(M. Del Castillo et al Clin. Infect. Dis. 2016, 63, 1490-1493). Seriousinfections were defined as infections requiring hospitalization orparenteral antimicrobials. Of 740 patients (898 courses of immunecheckpoint blockade), serious infection developed in 54 patients (7.3%).Nine patients (17%) were deemed to have died of an infection. Totalnumber of infections was 58, as some patients developed >1 infection.The majority of infections were bacterial in origin (˜80%; i.e.,bacterial infections: 80% of 7.3%: 5.8% of patients). Pneumonia andbloodstream infections were the two dominating bacterial infectiontypes.

Immune checkpoint-blocking drugs are associated with immune-relatedadverse effects (irAEs) related to the upregulated immune system. Thecomplications are managed with immunosuppressive drugs, such as steroids(immunosuppression is a risk factor for subsequent opportunisticinfections). Of the 740 patients, 46% received steroids during thecourse of treatment. Risk of serious infections was 13.5% in the cohortreceiving corticosteroids or infliximab (vs. 7.3% in the overallpopulation).

In yet another report, the emerging concern of infectious diseases inlung cancer patients receiving ICI therapy was investigated. Of 84 NSCLCpatients receiving nivolumab (a PD-1 inhibitor), 20 patients (23.8%)developed an infectious disease. Bacterial infections accounted for 75%of infections; i.e., bacterial infections in 18% of patients. Mostcommon type of bacterial infection was pneumonia. See K. Fujita et alEur. Resp. J. 2017, 50, OA1478.

The Gram-negative bacillus E. coli is one of the most common causes ofbacteraemia in patients with cancer. The all-cause 30-day mortality ratefor this pathogen is high (˜15%) (Y. E. Ha et al Int. J. Antimicr. Agen.2013, 42, 403-409). Published estimates of 30-day all-cause mortalityamong E. coli bacteraemia patients (cancer/non-cancer) vary from around10 to 35% (J. K. Abernethy et al Clin. Microbiol. Infect. 2015, 21,251.e1-251.e8), clearly highlighting the high burden associated withjust this pathogen. Overall, causative pathogens in bacteraemia areprimarily Gram-negative bacteria (65%), with E. coli (18.3%), P.aeruginosa (18.3%), and K pneumoniae (17.3%) being the most commonorganisms encountered; the three pathogens together account for 54% ofthe bacteraemia cases, or 85% of Gram-negative cases, according to astudy investigating >100 bacteraemia cases in cancer patients (G.Samonis et al Support Care Cancer 2013, 21, 2521-2526). In-hospitalmortality was 26.2% in this study. Comparable numbers can be foundelsewhere. For example, a study of neutropenic and non-neutropenic adultcancer patients with bloodstream infections investigated 399 cases ofbloodstream infections in 344 cancer patients: The largest causativepathogen group was Gram-negative bacilli (45%). Of the clinicalisolates, E. coli (35%) accounted for the most cases withinGram-negatives, followed by K. pneumoniae (20%) and P. aeruginosa (19%)(E. Velasco et al Eur. J. Clin. Microbiol. Infect. Dis. 2006, 25, 1-7).The three pathogens collectively account for 33% of the bacteraemiacases (or 74% of Gram-negative cases). The overall 30-day mortality ratewas 32% in this study. Two other reports looked at causative agents ofbloodstream infection in patients with solid tumours and also foundGram-negative bacteria to be the dominating pathogen type (47-55% of theinfections, across several hundred patients) (M. Marin et al Medicine2014, 93, 143-149; M. Anatoliotaki et al Infection 2004, 32, 65-71; seealso C. Gudiol et al Virulence 2016, 7, 298-308). In the larger of thetwo studies (with more robust numbers for individual pathogens), thethree main pathogens within the Gram-negative group were again E. coli(55%), P. aeruginosa (18%), and Klebsiella spp. (11%)—corresponding to92% of the Gram-negative cases, or 51% of the 528 total cases ofbloodstream infections studied.

The above data on specific causative infectious pathogens in cancerpatients are summarized in Table 5 below.

Thus, available data on bloodstream infections in cancer indicate thatGram-negative pathogens are involved in 45-65% of the infection cases,with three key pathogens—E. coli, K. pneumoniae, and P. aeruginosa—beingthe culprits in the vast majority of Gram-negative cases (73-92%).

The inventors, thus, formulated an oncologist's dilemma:

-   -   Reduction in efficacy of the cancer therapy is likely due to the        reduced microbiome diversity resulting from antibiotic therapy    -   At least ⅓ of patients on checkpoint inhibitors get serious and        life-threatening infections    -   Not treating these infections could result in death from the        infection (1-2 weeks)    -   Treatment with classic antibiotics leads to reduction in        progression free survival after 4 years from >40% to around 10%.    -   The choice is to treat the immediate need of a potentially fatal        infection (which must be addressed) at the risk of seriously        undermining the cancer therapy.

The inventors realised, therefore, that there is a need for methods thatcan treat a bacterial pathogenic infection in a different way thatminimizes compromise to the cancer therapy. The inventors realised thatthis need would also be useful in other therapy settings where themicrobiome composition can modulate therapy outcomes, eg, in transplantsettings.

Whilst not wishing to be bound by any particular theory, the inventorsbelieve that alleviating the detrimental effect of traditionalantibiotic therapy on overall survival in ICI patients using theinvention may, in some embodiments, translate to as much as a doublingof overall survival (or >12 months). Capturing a treatment effect ofseveral months in terms of median overall survival is a very substantialachievement in this space. In fact, an effect size of this order ofmagnitude is comparable to the outcomes reported for ICI trials (i.e.,where benefits usually are measured in months, not years). Additionally,PD-1/PD-L1 drugs are projected to dominate the ICI market. In 2023,PD-1/PD-L1 are projected to account for 94% of $46B USD global sales ofICIs (CTLA-4 blockers only account for 6%), source: “Landscape &Forecast: Immune Checkpoint Inhibitors”, Decision Resources, December2017. Thus, a need for improving treatment using immune checkpointinhibitors of PD-1 or PD-L1 is particularly pressing in medicine, and webelieve that the present invention finds particular benefit in thisrespect.

In an example, the method removes the need to administer a classicantibiotic, such as a broad-spectrum antibiotic (or any other onedisclosed herein). In another example, the invention reduces the amountor dosing frequency of a classic antibiotic, such as a broad-spectrumantibiotic (or any other one disclosed herein) that is administered tothe subject for treating the infection. For example, the subject can beadministered a low-dose broad-spectrum antibiotic (eg, 50, 40, 30, 20,10% or less of a conventional dose) whilst the guided nuclease cuttingis used, and thus treatment of the infection in this setting. Theinvention may be particularly beneficial for patients onimmunosuppressants, eg, for cancer patients, transplant patients orpatients suffering from a viral infection (eg, HIV (humanimmunodeficiency virus), CMV (cytomegalovirus) or RSV (respiratorysynctial virus) infection).

The term “broad-spectrum antibiotic” can refer to an antibiotic thatacts on the two major bacterial groups, gram-positive and gram-negative,or any antibiotic that acts against a wide range of disease-causingbacteria. These medications are used when a bacterial infection issuspected but the group of bacteria is unknown (also called empirictherapy) or when infection with multiple groups of bacteria issuspected. Although powerful, broad-spectrum antibiotics pose specificrisks, particularly the disruption of native, normal bacteria and thedevelopment of antimicrobial resistance. Examples of commonly usedbroad-spectrum antibiotics are Aminoglycosides (except forstreptomycin), Ampicillin, Amoxicillin, Amoxicillin, clavulanic acid(Augmentin), Carbapenems (e.g. imipenem), Piperacillin, tazobactam,Quinolones (e.g. ciprofloxacin), Tetracyclines, Chloramphenicol,Ticarcillin, Trimethoprim and sulfamethoxazole (Bactrim). In veterinarymedicine, examples are co-amoxiclav, (eg, in small animals), penicillin,streptomycin, oxytetracycline and potentiated sulfonamides.

Clauses:—

The invention, therefore, in one aspect provides the following Clausesthat are directed to the treatment of a pathogenic bacterial infectionusing a programmed nuclease.

-   -   1. A method for treating a pathogenic bacterial infection in a        human or animal subject caused by bacteria (first bacteria) of a        first species or strain, the method comprising selectively        killing first bacteria comprised by the subject by cutting a        target site comprised by the genomes of the first bacteria,        wherein the cutting is carried out using a programmable nuclease        that is programmed to cut the target site, wherein the subject        is suffering from a further disease or condition other than the        pathogenic bacterial infection and the method comprises        administering a therapy to the subject for treating or        preventing the further disease or condition, wherein the        nuclease treats the infection and the therapy is efficacious in        the presence of the programmed nuclease to treat or prevent the        disease or condition.

In an example, Clause 1 provides:—

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer;

Wherein

-   -   (a) The immunotherapy comprises administering to the patient an        anti-PD-1 antibody optionally selected from pembrolizumab (or        KEYTRUDA™) and nivolumab (or OPDIVO™); and    -   (b) The cancer is selected from metastatic melanoma; renal cell        carcinoma; bladder cancer; a solid tumour; non-small cell lung        cancer (NSCLC); forehead and neck squamous cell carcinoma        (HNSCC); Hodgkin's lymphoma; a cancer that overexpresses PD-L1        and no mutations in EGFR or in ALK; colorectal cancer and        hepatocellular carcinoma; and    -   (c) The first bacteria are selected from Pseudomonas aeruginosa,        Klebsiella pneumoniae, E. coli, Salmonella (eg, S. typhimurium),        Clostridium dificile, Staphylococcus (eg, S. aureus or S        epidermis), Streptococcus (eg, S. viridans or S. thermophilus),        Pneumococcus and Enterococcus bacteria.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer;

Wherein

-   -   (a) The immunotherapy comprises administering to the patient an        anti-PD-L1 antibody optionally selected from atezolimumab (or        TECENTRIQ™), avelumab (or BAVENCIO™) and durvalumab (or        IMFINZI™); and    -   (b) The cancer is selected from metastatic melanoma; renal cell        carcinoma; a solid tumour; non-small cell lung cancer (NSCLC);        forehead and neck squamous cell carcinoma (HNSCC); Merkel cell        carcinoma; Hodgkin's lymphoma; a cancer that overexpresses PD-L1        and no mutations in EGFR or in ALK; colorectal cancer and        hepatocellular carcinoma; and    -   (c) The first bacteria are selected from Pseudomonas aeruginosa,        Klebsiella pneumoniae, E. coli, Salmonella (eg, S. typhimurium),        Clostridium dificile, Staphylococcus (eg, S. aureus or S        epidermis), Streptococcus (eg, S. viridans or S. thermophilus),        Pneumococcus and Enterococcus bacteria.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer;

Wherein

-   -   (a) The immunotherapy comprises administering to the patient an        anti-CD52, antibody optionally alemtuzumab (or CAMPATH™); and    -   (b) The cancer is B-cell chronic lymphocytic leukemia (CLL); and    -   (c) The first bacteria are selected from Pseudomonas aeruginosa,        Klebsiella pneumoniae, E. coli, Salmonella (eg, S. typhimurium),        Clostridium dificile, Staphylococcus (eg, S. aureus or S        epidermis), Streptococcus (eg, S. viridans or S. thermophilus),        Pneumococcus and Enterococcus bacteria.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer;

Wherein

-   -   (a) The immunotherapy comprises administering to the patient an        anti-CD20 antibody, optionally ofatumumab (or ARZERRA™) or        rituximab (or RITUXAN™); and    -   (b) The cancer is B-cell chronic lymphocytic leukemia (CLL) (eg,        refractory CLL) or non-Hodgkin lymphoma; and    -   (c) The first bacteria are selected from Pseudomonas aeruginosa,        Klebsiella pneumoniae, E. coli, Salmonella (eg, S. typhimurium),        Clostridium dificile, Staphylococcus (eg, S. aureus or S        epidermis), Streptococcus (eg, S. viridans or S. thermophilus),        Pneumococcus and Enterococcus bacteria.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer;

Wherein

-   -   (a) The immunotherapy comprises administering to the patient an        anti-KIR antibody, optionally lirilumab; and    -   (b) The cancer is optionally acute myeloid leukaemia or squamous        cell carcinoma of the head and neck (SCCHN); and    -   (c) The first bacteria are selected from Pseudomonas aeruginosa,        Klebsiella pneumoniae, E. coli, Salmonella (eg, S. typhimurium),        Clostridium dificile, Staphylococcus (eg, S. aureus or S.        epidermis), Streptococcus (eg, S. viridans or S. thermophilus),        Pneumococcus and Enterococcus bacteria.

A method for treating a pathogenic bacterial infection in a cancerpatient caused by bacteria (first bacteria) of a first species orstrain, the method comprising selectively killing first bacteriacomprised by the subject by cutting a target site comprised by thegenomes of the first bacteria, wherein the cutting is carried out usinga Cas nuclease that is programmed by guide RNA to cut the target site,wherein the method comprises administering an immunotherapy to thesubject for treating cancer in the patient, wherein the nuclease treatsthe infection and the immunotherapy is efficacious in the presence ofthe programmed nuclease to treat the cancer;

Wherein

-   -   (a) The immunotherapy comprises administering to the patient an        anti-CD19 CAR-T optionally selected from axicabtagene ciloleucel        (or YESCARTA™) and tisagenlecleucel (or KYMRIAH™); and    -   (b) The cancer is selected from a B-cell lymphoma (eg,        non-Hodgkin's lymphoma (NHL); diffuse large B-cell lymphoma        (DLBCL); primary mediastinal large B-cell lymphoma; or high        grade B-cell lymphoma); B-cell acute lymphoblastic leukaemia        (ALL); or central nervous system lymphoma; and    -   (c) The first bacteria are selected from Pseudomonas aeruginosa,        Klebsiella pneumoniae, E. coli, Salmonella (eg, S. typhimurium),        Clostridium dificile, Staphylococcus (eg, S. aureus or S        epidermis), Streptococcus (eg, S. viridans or S. thermophilus),        Pneumococcus and Enterococcus bacteria.

Alternatively, the CAR-T is an anti-CD30, CD38 or CD22 CAR-T. In anexample the cancer is large B-cell lymphoma after at least two otherkinds of treatment failed. In an example the cancer is high grade B-celllymphoma and DLBCL arising from follicular lymphoma. In an example thecancer is relapsing/remitting B cell acute lymphoblastic leukaemia. Inan example the cancer is primary central nervous system lymphoma.

In an example, the nuclease treats the infection without causingreduction in efficacy of the therapy. In an embodiment, “without causingreduction in efficacy of the therapy” means the efficacy of the therapycompared to a reduction caused in patients by the administration of abroad-spectrum antibiotic (or an antibiotic disclosed herein) that killsa plurality of different species, wherein the plurality comprises thefirst species. In an embodiment, “without causing reduction in efficacyof the therapy” means the efficacy of the therapy is reduced by no morethan 70, 80, 90 or 95% compared to administration of the therapy in theabsence of treatment of the pathogenic bacterial infection (or comparedto therapy as typically achieved in patients suffering from the diseaseor condition and receiving said therapy therefor). This may be assessed,for example, by determining the duration of progression-free survival ofthe subject or treatment of the disease or condition, or overallsurvival of the subject; and/or by determining a reduction in one ormore symptoms of the disease or condition.

In an example, the infection is treated completely or substantiallycompletely. In another example, the infection is reduced (eg, by atleast 80, 90 or 95% as determined by a marker of the infection or asymptom thereof). A marker may, for example, be CFUs of bacteria of thefirst species or strain per ml of a blood sample taken from the patientafter the method has been carried out, eg, within 24 hours of thatmethod being carried out, eg, from 1-12 hours or 1-24 hours aftercarrying out the method or from 1-12 hours or 1-24 hours afteradministering a RNA or DNA encoding the RNA to programme the nuclease inthe subject. For example, the RNA is a guide RNA and the nuclease is Cas(eg, a Cas3 or a Cas9). The reduction may be compared to a sample takenfrom the subject immediately prior to the commencement of the method.Alternatively, the sample may be a stool, saliva or urine sample.

In an example, the invention increases overall survival rate in a humansubject (compared to median overall survival rate in humans sufferingfrom the same cancer and receiving the same cancer therapy treatment(eg, administration of the same immune checkpoint inhibitor, such asnivolumab, pembrolizumab or another antibody disclosed herein)). In anexample any composition, or other product of the invention herein isprovided for use in such method of treatment.

In an example, the method is practised on a population of human subjectsand the median overall survival rate for the population is 120-250% (eg,150-200%) of the median overall survival rate in humans suffering fromthe same cancer and receiving the same cancer therapy treatment (eg,administration of the same immune checkpoint inhibitor, such asnivolumab, pembrolizumab or another antibody disclosed herein). In anexample any composition, or other product of the invention herein isprovided for use in such method of treatment.

A “pathogenic bacterial infection” is a health-threatening infection ofthe subject, for example, a life-threatening infection. In anembodiment, a pathogenic bacterial infection is an infection requiringhospitalization or parenteral antimicrobials. The infection may be anacute bacterial infection, such as a systemic infection or a localisedinfection. Bacterial pathogens often cause infection in specific areasof the body. Others are generalists. A pathogenic bacterial infection iscontrasted with an infection of commensal bacteria, such as commensalgut bacteria; in this case the bacteria do not cause an immediatehealth- or life-threatening situation.

The infection (or symptom thereof) can be any of the following:—

-   -   Bacterial vaginosis: this is caused by bacteria that change the        vaginal microbiota caused by an overgrowth of bacteria that        crowd out the Lactobacilli species that maintain healthy vaginal        microbial populations.    -   Bacterial meningitis: this is a bacterial inflammation of the        meninges, that is, the protective membranes covering the brain        and spinal cord.    -   Bacterial pneumonia: this is a bacterial infection of the lungs.    -   Urinary tract infection: this is predominantly caused by        bacteria. Symptoms include the strong and frequent sensation or        urge to urinate, pain during urination, and urine that is        cloudy. The main causal agent is Escherichia coli. Bacteria can        ascend into the bladder or kidney and causing cystitis and        nephritis.    -   Bacterial gastroenteritis: this is caused by enteric, pathogenic        bacteria. These pathogenic species are usually distinct from the        usually harmless bacteria of the normal gut flora. But a        different strain of the same species may be pathogenic.    -   Bacterial skin infections: these include:        -   Impetigo, which is a highly contagious bacterial skin            infection commonly seen in children. It is caused by            Staphylococcus aureus, and Streptococcus pyogenes.        -   Erysipelas, which is an acute streptococcus bacterial            infection of the deeper skin layers that spreads via with            lymphatic system.        -   Cellulitis, which is a diffuse inflammation of connective            tissue with severe inflammation of dermal and subcutaneous            layers of the skin. Cellulitis can be caused by normal skin            flora or by contagious contact, and usually occurs through            open skin, cuts, blisters, cracks in the skin, insect bites,            animal bites, burns, surgical wounds, intravenous drug            injection, or sites of intravenous catheter insertion. In            most cases it is the skin on the face or lower legs that is            affected, though cellulitis can occur in other tissues.

In an example, the first bacteria are Streptococcus and the patient issuffering from chest infection, cellulitis or tonsillitis. In anexample, the first bacteria are Enterococcus and the patient issuffering from bladder infection or septicaemia. In an example, thefirst bacteria are Pseudomonas aeruginosa and the patient is sufferingfrom diarrhoea. In an example, the first bacteria are E. coli and thepatient is suffering from diarrhoea.

-   -   2. The method of Clause 1, wherein the subject is a cancer        patient and the therapy is a cancer therapy.    -   3. The method of Clause 2, wherein the therapy comprises        administration of a haematopoietic stem cell transplant,        chemotherapeutic agent, immune checkpoint inhibitor, immune        checkpoint agonist or an immune cell (eg, T-cell and/or NK cell)        enhancer; adoptive cell therapy (eg, CAR-T therapy); radiation        or surgery.

In an example, the therapy is immunotherapy. Examples of suitableimmunotherapy are administration of adoptive cell therapy (eg, CAR-Ttherapy), an immune checkpoint inhibitor, an immune checkpoint agonistor an immune cell (eg, T-cell and/or NK cell) enhancer. For example,administration of an anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3, 0X40, CD28,BTLA, CD137, CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR, TIGIT or SIRPaantibody, such as administration of an antibody selected from ipilimumab(or YERVOY™), tremelimumab, nivolumab (or OPDIVO™) pembrolizumab (orKEYTRUDA™), pidilizumab, BMS-936559, durvalumab and atezolizumab, or aCAR-T therapy such as axicabtagene ciloleucel (Yescarta™) ortisagenlecleucel (Kymriah™).

In an example, the immune enhancer comprises an interleukin-2 (IL-2) orfragment or deletion mutant thereof.

In an example, the surgery comprises the removal of necrotic orcancerous tissue.

In an example, the chemotherapy comprises administration of aplatinum-containing chemotherapy drug. In an example, the chemotherapycomprises administration of gefitinib.

In an example, the therapy comprises administering Cyclophosphamide,methotrexate and 5-fluorouracil (CMF); or doxorubicin andcyclophosphamide (AC); docetaxel, doxorubicin and cyclophosphamide(TAC); or doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD); ormustine, vincristine, procarbazine and prednisolone (MOPP);cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP);bleomycin, etoposide and cisplatin (BEP); epirubicin, cisplatin and5-fluorouracil (ECF); or epirubicin, cisplatin and capecitabine (ECX);methotrexate, vincristine, doxorubicin and cisplatin (MVAC);cyclophosphamide, doxorubicin and vincristine (CAV); or 5-fluorouracil,folinic acid and oxaliplatin (FOLFOX).

In an example, the cancer is breast cancer and the therapy comprisesadministering CMF or AC. In an example, the cancer is Hodgkin's lymphomaand the therapy comprises administering TAC, ABVD or MOPP. In anexample, the cancer is Non-Hodgkin's lymphoma and the therapy comprisesadministering CHOP. In an example, the cancer is germ cell cancer andthe therapy comprises administering BEP. In an example, the cancer isstomach cancer and the therapy comprises administering ECF or ECX. In anexample, the cancer is bladder cancer and the therapy comprisesadministering MVAC. In an example, the cancer is lung cancer and thetherapy comprises administering CAV. In an example, the cancer iscolorectal cancer and the therapy comprises administering FOLFOX.

-   -   4. The method of Clause 3, wherein the therapy is an immune        checkpoint inhibitor antibody.

Optionally the antibody is an anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3,0X40, CD28, BTLA, CD137, CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR,TIGIT or SIRPa antibody. In an example, the antibody is an anti-PD-1antibody. In an example, the antibody is an anti-PD-L1 antibody. In anexample, the antibody is an anti-CTLA4 antibody.

-   -   5. The method of Clause 3, wherein the therapy is administration        of an antibody selected from ipilimumab (or YERVOY™),        tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or        KEYTRUDA™), pidilizumab, BMS-936559, durvalumab and        atezolizumab.

Optionally, the antibody (eg, anti-PD-L1 antibody) is administered withan anti-CTLA4 antibody (eg, ipilimumab or tremelimumab).

In an example, the an anti-PD-1 antibody herein is selected fromnivolumab, pembrolizumab, pidillizumab, OPDIVO®, KEYTRUDA®, AMP-514,REGN2810, CT-011, BMS 936559, MPDL3280A and AMP-224.

In an example, the an anti-CTLA4 antibody herein is selected fromtremelimumab, YERVOY® and ipilimumab.

In an example the therapy is administration of an anti-KIR antibody, eg,lirilumab.

In an example, the checkpoint inhibitor is selected from an inhibitor ofCTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR.In certain aspects, the immune checkpoint inhibitor is a humanprogrammed cell death 1 (PD-1) axis-binding antagonist. In some aspects,the PD-1 axis-binding antagonist is selected from the group consistingof a PD-1 binding antagonist, a PD-L1-binding antagonist and aPD-L2-binding antagonist. In certain aspects, the PD-1 axis-bindingantagonist is a PD-1-binding antagonist. In some aspects, thePD-1-binding antagonist inhibits the binding of PD-1 to PD-L1 and/orPD-L2.

In some embodiments, the immune checkpoint inhibitor is a PD-L1antagonist such as durvalumab, also known as MEDI4736, atezolizumab,also known as MPDL3280A, or avelumab, also known as MSB00010118C. Incertain aspects, the immune checkpoint inhibitor is a PD-L2 antagonistsuch as rHIgM12B7. In some aspects, the immune checkpoint inhibitor is aLAG-3 antagonist such as IMP321 or BMS-986016. The immune checkpointinhibitor may be an adenosine Ata receptor (A2aR) antagonist such asPBF-509.

In some embodiments, the antibody described herein (such as an anti-PD-1antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody) furthercomprises a human or murine constant region. In a still further aspect,the human constant region is selected from the group consisting of IgG1,IgG2, IgG2, IgG3, and IgG4. In a still further specific aspect, thehuman constant region is IgG1. In a still further aspect, the murineconstant region is selected from the group consisting of IgG1, IgG2A,IgG2B, and IgG3. In a still further specific aspect, the antibody hasreduced or minimal effector function. In a still further specificaspect, the minimal effector function results from production inprokaryotic, CHO, Cos or HEK cells. In a still further specific aspectthe minimal effector function results from an “effector-less Fcmutation” or aglycosylation.

For example, the therapy comprises a haemopoietic stem cell transplant,eg, a bone marrow transplant (such as when the patient is a cancerpatient, eg, a blood cancer or leukaemia patient).

For example, the therapy comprises a stem cell transplant, a skin graft,or an organ transplant, eg, a heart, liver, kidney or lung transplant.

-   -   6. The method of Clause 1 or 2, wherein the therapy is a tissue,        organ or cell transplant.    -   7. The method of any preceding Clause, wherein the treatment of        the bacterial infection is carried out simultaneously with the        administration of the therapy to the subject.    -   8. The method of any one of Clauses 1 to 6, wherein the        treatment of the bacterial infection is carried out immediately        before administering the therapy to the subject.

In an example, the treatment of the bacterial infection is carried outno more than 7, 6, 5, 4, 3, 2, or 1 day, or 24, 12, 6, 5, 4, 3, 2, 1 or0.5 hours before the therapy of the further disease or condition. In anexample, the treatment of the bacterial infection is carried out no morethan 7, 6, 5, 4, 3, 2, or 1 day, or 24, 12, 6, 5, 4, 3, 2, 1 or 0.5hours after the therapy of the further disease or condition.

The treatment of the infection and the administration of the therapy maybe carried out simultaneously or sequentially.

-   -   9. The method of any one of Clauses 1 to 6, wherein the        treatment of the bacterial infection is carried out immediately        after administering the therapy to the subject.    -   10. The method of any preceding Clause, wherein the method        comprises administering to the subject a RNA (eg, a gRNA) or a        nucleic acid that encodes an RNA for expression of the RNA in        the subject, wherein the RNA complexes with the nuclease to        program the nuclease to cut the target site in first bacteria        comprised by the subject, thereby killing the first bacteria.

The RNA or nucleic acid is, for example, administered to the subject orpatient orally, by IV injection, by subcutaneous injection or byinhalation.

-   -   11. The method of any preceding Clause, comprising administering        a vector (eg, phage or plasmids) to the subject, wherein the        vector encodes the programmable nuclease.

The nuclease is, for example, administered to the subject or patientorally, by IV injection, by subcutaneous injection or by inhalation.

-   -   12. The method of any one of Clauses 1 to 10, wherein the        programmable nuclease is an endogenous nuclease (eg, Cas        nuclease) of the first cells.    -   13. The method of any preceding Clause, wherein the efficacy of        the therapy in the presence of the programmed nuclease is        greater than the efficacy of the therapy in the presence of a        broad-spectrum antibiotic.

In an example, the efficacy being greater is assessed by determining theduration of progression-free survival or treatment of the disease orcondition; and/or by determining a reduction in one or more symptoms ofthe disease or condition. For example, this determination is compared toan analogous determination in a patient suffering from the disease orcondition as well as the bacterial infection and being treated with thetherapy and the antibiotic (rather than the nuclease killing of firstbacteria as per the invention).

-   -   14. The method of any preceding Clause, wherein the efficacy of        the therapy in the presence of the programmed nuclease is        greater than the efficacy of the therapy in the presence of an        antibiotic selected from methicillin, vancomycin, linezolid,        daptomycin, quinupristin, dalfopristin; teicoplanin;        cephalosporin; carbapenem; fluoroquinolone; aminoglycoside;        colistin; erythromycin; clindamycin; beta-lactam; macrolide;        amoxicillin; azithromycin; penicillin; ceftriaxone;        azithromycin; ciprofloxacin; isoniazid (INH); rifampicin (RMP);        amikacin; kanamycin; capreomycin; trimethoprim; itrofurantoin;        cefalexin; amoxicillin; metronidazole (MTZ); cefixime;        tetracycline; and meropenem.    -   15. The method of any preceding Clause, wherein the first        bacteria is selected from (i) Staphylococcus aureus that is        resistant to an antibiotic selected from methicillin,        vancomycin, linezolid, daptomycin, quinupristin, dalfopristin        and teicoplanin; (ii) Pseudomonas aeuroginosa that is resistant        to an antibiotic selected from cephalosporins, carbapenems,        fluoroquinolones, aminoglycosides and colistin; (iii) Klebsiella        species that is resistant to carbapenem; (iv) Streptococcus        species that is resistant to an antibiotic selected from        erythromycin, clindamycin, beta-lactam, macrolide, amoxicillin,        azithromycin and penicillin; (v) Salmonella species that is        resistant to an antibiotic selected from ceftriaxone,        azithromycin and ciprofloxacin; (vi) Shigella species that is        resistant to ciprofloxacin or azithromycin; (vii) Mycobacterium        tuberculosis that is resistant to an antibiotic selected from        Resistance to isoniazid (INH), rifampicin (RMP),        fluoroquinolone, amikacin, kanamycin, capreomycin and        azithromycin; (viii) Enterococcus species that is resistant to        vancomycin; (ix) Enterobacteriaceae species that is resistant to        an antibiotic selected from cephalosporin and carbapenem; (x) E.        coli that is resistant to an antibiotic selected from        trimethoprim, itrofurantoin, cefalexin and amoxicillin; (xi)        Clostridium species that is resistant to metronidazole (MTZ),        fluoroquinolone or carbapenem; (xii) Neisseria gonnorrhoea that        is resistant to an antibiotic selected from cefixime,        ceftriaxone, azithromycin and tetracycline; (xiii) Acinetobacter        baumannii that is resistant to an antibiotic selected from        beta-lactam, meropenem and carbapenem; and (xiv) Campylobacter        species that is resistant to ciprofloxacin or azithromycin.    -   16. The method of any preceding Clause, wherein the treatment of        the infection treats or prevents in the subject a condition        selected from vaginosis, meningitis, pneumonia, urinary tract        infection, cystitis, nephritis, gastroenteritis, a skin        infection, impetigo, erysipelas, dental infection and        cellulitis.    -   17. The method of any preceding Clause, wherein the treatment of        the infection treats or prevents septicaemia or sepsis in the        subject.

In an example, the infection is a bloodstream infection.

-   -   18. The method of any preceding Clause, wherein the further        disease or condition is a cancer; autoimmune disease or        condition; viral infection or GI tract disease or condition. In        an example, the cancer is metastatic. In an example, the cancer        is melanoma. In an example, the cancer is a solid tumour with        mismatch repair deficiency or microsatellite instability. In an        example, the cancer is NSCLC. In an example, the cancer is        HNSCC. In an example, the cancer is Hodgkin's lymphoma. In an        example, the cancer is urothelial cancer. In an example, the        cancer is lung cancer. In an example, the cancer is head and        neck cancer. In an example, the cancer is head cancer. In an        example, the cancer is neck cancer. In an example, the viral        infection is a HIV, CMV or RSV infection.    -   19. The method of any preceding Clause, wherein the subject        comprises bacteria (second bacteria) of one or more strains or        species that are different to the first strain or species,        wherein the genomes of the second bacteria do not comprise the        target site, wherein the genomes of the second bacteria are not        cut by the programmed nuclease in the subject, whereby second        bacteria survive in the presence of the programmed nuclease in        the patient; and wherein the therapy is efficacious in the        presence of the second bacteria.    -   20. The method of Clause 19, wherein reduction in the second        bacteria in patients (eg, in the gut microbiome) is associated        with reduced efficacy of the therapy.

Optionally, the therapy is efficacious in the presence of the secondbacteria in the gut of the subject.

Optionally, the first and/or second bacteria are present in the gut ofthe subject immediately prior to carrying out the method.

Optionally, the first and/or second bacteria are present in the blood ofthe subject immediately prior to carrying out the method.

Optionally, the first bacteria are present in the blood of the subjectand the second bacteria are present in the gut of the subjectimmediately prior to carrying out the method.

Optionally, the first bacteria are present in the gut of the subject andthe second bacteria are present in the blood of the subject immediatelyprior to carrying out the method.

Optionally, first bacteria in the blood of the subject is killed.

Optionally, the bacteria are gram positive bacteria. Optionally, thebacteria are gram negative bacteria.

Optionally, the first and second bacteria are capable of being killed bythe same antibiotic. Optionally, the method does not compriseadministering the antibiotic to the subject. In an example, theantibiotic is selected from methicillin, vancomycin, linezolid,daptomycin, quinupristin, dalfopristin; teicoplanin; cephalosporin;carbapenem; fluoroquinolone; aminoglycoside; colistin; erythromycin;clindamycin; beta-lactam; macrolide; amoxicillin; azithromycin;penicillin; ceftriaxone; azithromycin; ciprofloxacin; isoniazid (INH);rifampicin (RMP); amikacin; kanamycin; capreomycin; trimethoprim;itrofurantoin; cefalexin; amoxicillin; metronidazole (MTZ); cefixime;tetracycline; and meropenem. In an example, the antibiotic is selectedfrom Aminoglycosides, Ampicillin, Amoxicillin, Amoxicillin or clavulanicacid, Carbapenems (e.g. imipenem), Piperacillin or tazobactam,Quinolones (e.g. ciprofloxacin), Tetracyclines, Chloramphenicol,Ticarcillin, Trimethoprim or sulfamethoxazole, penicillin, streptomycin,oxytetracycline and potentiated sulfonamides. In an example, the firstbacteria are resistant to an antibiotic selected from Aminoglycosides,Ampicillin, Amoxicillin, Amoxicillin or clavulanic acid, Carbapenems(e.g. imipenem), Piperacillin or tazobactam, Quinolones (e.g.ciprofloxacin), Tetracyclines, Chloramphenicol, Ticarcillin,Trimethoprim or sulfamethoxazole, penicillin, streptomycin,oxytetracycline and potentiated sulfonamides. In an alternative, theantibiotic is selected from a beta-lactam, fluoroquinolone andmacrolide.

Optionally, the first and second bacteria are bacteria of the samespecies, but are different strains of the species.

Optionally, the first and second bacteria are bacteria of the samegenus, but are bacteria of different species of the genus.

Optionally, the first and second bacteria are bacteria of the samefamily, but are bacteria of different genera of the family.

Optionally, the first and second bacteria are gram positive bacteria.

Optionally, the first and second bacteria are gram-negative bacteria.

Optionally, the therapy is efficacious in the presence of the secondbacteria.

Optionally, reduction in the second bacteria in patients is associatedwith reduced efficacy of the therapy. Optionally, reduction in thesecond bacteria in patients reduces efficacy of the therapy.

Optionally, the presence of the second bacteria in patients isassociated with enhanced efficacy of the therapy. Optionally, thepresence of the second bacteria in patients enhances efficacy of thetherapy. For example, enhanced efficiency is efficiency compared totherapy in the absence or a reduced presence of the second bacteria,such as in the presence of an antibiotic that kills the second bacteria.

In an example, the therapy is efficacious in the presence of the secondbacteria, wherein the disease or condition (or a symptom thereof) isreduced in the subject by at least 20, 30, 40, 50, 60, 70, 80, 90 or95%. In an example, the therapy is efficacious in the presence of thesecond bacteria, wherein the progression of the disease or condition (ora symptom thereof) is reduced in the subject by at least 20, 30, 40, 50,60, 70, 80, 90 or 95%. In an example, the therapy is efficacious in thepresence of the second bacteria, wherein disease-free progression of thedisease or condition (or a symptom thereof) is reduced in the subject byat least 20, 30, 40, 50, 60, 70, 80, 90 or 95%. In an example, thetherapy is efficacious in the presence of the second bacteria, whereinthe duration of the disease or condition (or a symptom thereof) isreduced in the subject by at least 20, 30, 40, 50, 60, 70, 80, 90 or95%. In an example, the therapy is efficacious in the presence of thesecond bacteria, wherein the severity of the disease or condition (or asymptom thereof) is reduced in the subject by at least 20, 30, 40, 50,60, 70, 80, 90 or 95%. In an example, the therapy is efficacious in thepresence of the second bacteria, wherein the disease or condition (or asymptom thereof) is reduced for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 21 or 28 days or for at least 1, 2, 3, 4 5, 6 or 12months in the patient by at least 20, 30, 40, 50, 60, 70, 80, 90 or 95%.In an example, the therapy is efficacious in the presence of the secondbacteria, wherein the disease or condition (or a symptom thereof) istreated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21or 28 days or for at least 1, 2, 3, 4 5, 6 or 12 months in the patientby at least 20, 30, 40, 50, 60, 70, 80, 90 or 95%. In an example, thetherapy is efficacious in the presence of the second bacteria, whereinthe disease or condition (or a symptom thereof) is undetectable for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21 or 28 days orfor at least 1, 2, 3, 4 5, 6 or 12 months in the patient by at least 20,30, 40, 50, 60, 70, 80, 90 or 95%.

-   -   21. The method of Clause 19 or 20, wherein the second bacteria        are selected from the group consisting of Akkermansia,        Alistipes, Bacteroides, Barnesiella, Bifidobacterium,        Clostridium, Collinsella, Enterococcus, Fusobacterium,        Lactobacillus, Propionibacterium, Ruminococcus, Segmented        filamentous bacteria (SFB); Veillonella, Prevotella, Escherichia        and Streptococcus bacteria.

In an example the second bacteria bacteria that produce short chainfatty acids (eg, butyrate-producing bacteria). In particular aspects,the species of bacteria produce butyrate. For example, the secondbacteria are Clostridiales. The Clostridiales bacteria may besubstantially or include bacteria in spore form. In particular aspects,the second bacteria are of the family Ruminococcaceae,Christensenellaceae, Clostridiaceae or Coriobacteriacease. In someembodiments, the Clostridiales (eg, Clostridium) bacteria comprise afirst family and a second family. In some embodiments, the first familyis selected from the group consisting of Ruminococcaceae,Christensenellaceae, Clostridiaceae and Coriobacteriacease, and thesecond family is not identical to the first family. In an example, thesecond bacteria are Faecalibacterium prausnitzii, Ruminococcus albus,Ruminococcus bromii, Ruminococcus callidus, Ruminococcus flavefaciens,Ruminococcus champanellensis, Ruminococcus faecis, Ruminococcusgauvreauii, Ruminococcus gnavus, Ruminococcus hansenii, Ruminococcushydrogenotrophicus, Ruminococcus lactaris, Ruminococcus luti,Ruminococcus obeum, Ruminococcus palustris, Ruminococcus pasteurii,Ruminococcus productus, Ruminococcus schinkii, Ruminococcus torques,Subdoligranulum variabile, Butyrivibrio fibrisolvens, Roseburiaintestinalis, Anaerostipes caccae, Blautia obeum, Eubacterium nodatum orEubacterium oxidoreducens. In particular aspects, the second bacteriaare Faecalibacterium prausnitzii. In an example the second bacteria areFirmicutes.

In certain embodiments, the first bacteria are Bacteroidia orPrevotellaceae, eg, Bacteroidetes or Bacteroides.

In an embodiment, the treatment results in or maintains a microbiome(eg, gut and/or blood microbiome) of the subject, which is beneficialfor the immne checkpoint inhibitionor other therapy. In an example, themicrobiome comprises a high relative abundance of one or more bacterialspecies from the phylum Firmicutes, class Clostridia, orderClostridiales, family Ruminococcaceae, genus Ruminococcus, genusHydrogenoanaerobacterium, genus Faecalibacterium, phylum Actinobacteria,class Coriobacteriia, order Coriaobacteriales, family Coriobacteriaceae,domain Archaea, phylum Cyanobacteria, phylum Euryarchaeota or famlyChristensenellaceae. Additionally or alternatively, the microbiomecomprises a low relative abundance of bacteria from the genus Dialister,family Veillonellaceae, phylum Bacteroidetes, class Bacteroida, orderBacteroidales or family Prevotellaceae. Accordingly, a favorablemicrobial profile would have a higher relative abundance of one or morebacterial species from the phylum Firmicutes, class Clostridia, orderClostridiales, famiy Ruminococcaceae, genus Ruminococcus, genusHydrogenoanaerobacterium, phylum Actinobacteria, class Coriobacteria,order Coriaobacteriales, family Coriobacteriaceae, domain Archaea,phylum Cyanobacteria, phylum Euryarchaeota or familyChristensenellaceae, and/or has a decreased abundance of one or morebacterial species from genus Dialister, family Veillonellaceae, phylumBacteroidetes, class Bacteroida, order Bacteroidales and/or familyPrevotellaceae.

For example, the microbiome comprises a higher relative abundance ofFirmicutes compared to Bacteroidetes, Bacteroida, Bacteroidales orPrevotellaceae. For example, the microbiome comprises a higher relativeabundance of Firmicutes compared to Bacteroidetes, Bacteroida,Bacteroidales and Prevotellaceae.

Optionally, the second bacteria are selected from the group consistingof Akkermansia muciniphila; Alistipes shahii; Bacteroides fragilis;Bacteroides uniformis; Barnesiella intestinihominis; Bacteroides dorei;Bifidobacterium adolescentis; Bifidobacterium breve; Bifidobacteriumlongum; Clostridium orbiscindens; Clostridium novyi; Clostridiumperfringens; Collinsella aerofaciens; Enterococcus hirae; Fusobacteriumnucleatum; Lactobacillus casei Shirota; L. casei A047; Lactobacillusrhamnosus; Propionibacterium granulosum; Ruminococcus gnavus; Segmentedfilamentous bacteria (SFB); Veillonella; Lactobacilli; Bacteroides;Clostridia; Prevotella; E. coli Nissle; Lactobacillus plantarum;Lactobacillus delbrueckii (eg, subsp. Bulgaricus); Lactobacillusparacasei; Lactobacillus acidophilus; Bifidobacterium infantis; andStreptococcus salivarius (eg, subsp. Thermophilus). See “The microbiomein cancer immunotherapy: Diagnostic tools and therapeutic strategies”;Laurence Zitvogel et al; Science 23 Mar. 2018: Vol. 359, Issue 6382, pp.1366-1370; DOI: 10.1126/science.aar6918.

In an example, the second bacteria are commensal bacteria in humans.

In an example, the first bacteria are comprised by gut microbiota, skinmicrobiota, oral cavity microbiota, throat microbiota, hair microbiota,armpit microbiota, vaginal microbiota, rectal microbiota, analmicrobiota, ocular microbiota, nasal microbiota, tongue microbiota, lungmicrobiota, liver microbiota, kidney microbiota, genital microbiota,penile microbiota, scrotal microbiota, mammary gland microbiota, earmicrobiota, urethra microbiota, labial microbiota, organ microbiota ordental microbiota.

In an example, the second bacteria are comprised by gut microbiota, skinmicrobiota, oral cavity microbiota, throat microbiota, hair microbiota,armpit microbiota, vaginal microbiota, rectal microbiota, analmicrobiota, ocular microbiota, nasal microbiota, tongue microbiota, lungmicrobiota, liver microbiota, kidney microbiota, genital microbiota,penile microbiota, scrotal microbiota, mammary gland microbiota, earmicrobiota, urethra microbiota, labial microbiota, organ microbiota ordental microbiota.

In an example, the first and/or second bacteria are blood-bornebacteria.

-   -   22. The method of any preceding Clause, wherein the first        bacteria are selected from the group consisting Staphylococcus,        Streptococcus, Enterococcus, Helicobacter, Legionella,        Heamophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella,        Pseudomonas or Stenotrophomonas bacteria.

H. pylori has been implicated in gastric cancer and gastric ulcers.Thus, in an example, the first bacteria are H. pylori and optionally thedisease is a cancer, such as gastric cancer. In an embodiment, thetherapy is chemotherapy or therapy with an immune checkpoint inhibitor(eg, an antibody). In an example, the first bacteria are H. pylori andthe disease is gastric ulcer(s). In an embodiment, triple therapy forgastric ulcers is administered to the subject.

In an example, the first bacteria are Gram-negative bacteria andoptionally the infection is a blood infection. In an example, the firstbacteria are selected from E. coli, P. aeruginosa and K. pneumoniae, andoptionally the infection is a blood infection.

-   -   23. The method of Clause 22, wherein the first bacteria are        selected from the group consisting of E. coli (eg, EHEC E.        coli), C. dificile, V. cholera, Staphylococcus (eg, S. aureus or        MRSA), Streptococcus pyogenes, Helicobacter pylori,        Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa and        Klebsiella pneumoniae bacteria.

In an example, the subject has been administered an immunosuppressantdrug, or is on a course of an immunosuppressant drug, eg, a steroid,such as a corticosteroid.

-   -   24. A programmable nuclease for use in the method of any        preceding Clause.    -   25. The method or nuclease of any preceding Clause, wherein the        nuclease is a Cas nuclease (eg, a Cas 3 or 9), a meganuclease, a        TALEN (Transcription activator-like effector nuclease) or zinc        finger nuclease.    -   26. A CRISPR/Cas system comprising a nuclease according to        Clause 24 or 25 for use in the method of any one of Clauses 1 to        23, wherein the nuclease is a Cas nuclease (eg, a Cas 3 or 9)        and the system comprises one or more guide RNAs (gRNAs) or DNA        encoding one or more guide RNAs, wherein each guide RNA is        capable of programming the Cas nuclease to cut a target site        comprised by the genomes of first bacteria.    -   27. A guide RNA or a DNA encoding a guide RNA for use in the        system of Clause 26.    -   28. A guide RNA or a DNA encoding a guide RNA for use in the        method of treating a pathogenic bacterial infection according to        any one of Clauses 1 to 23, wherein the guide RNA is capable of        programming the nuclease, wherein the nuclease is a Cas nuclease        (eg, a Cas9, Cas3, Cas13, CasX, CasY or Cpf1 nuclease).    -   29. A nucleic acid vector comprising the guide RNA or DNA        recited in any one of Clauses 26 to 28.    -   30. A nucleic acid vector encoding the nuclease of Clause 24 or        25 and optionally the guide RNA of Clause 29.    -   31. The vector of Clause 29 or 30 wherein the vector is a phage,        phagemid plasmid (eg, conjugative plasmid) or transposon.

The phage are capable of infecting first bacteria and the phagemids arecapable of producing such phage in the presence of a helper phage.

-   -   32. A pharmaceutical composition comprising a first nucleic acid        vector (or a plurality thereof) encoding the nuclease of Clause        24 or 25 and a second nucleic acid vector (or a plurality        thereof) encoding the guide RNA of Clause 29, the composition        further comprising a pharmaceutically acceptable diluent,        excipient or carrier.    -   33. A pharmaceutical composition comprising the CRISPR/Cas        system of claim 26 and a pharmaceutically acceptable diluent,        excipient or carrier.    -   34. A pharmaceutical composition comprising the vector of claim        31 and a pharmaceutically acceptable diluent, excipient or        carrier.

Preventing a disease or condition herein may, for example, be reducingthe risk of the disease or condition in the subject or patient.

In an alternative, instead of first bacteria, the infection is caused byfirst archaea and in this embodiment all of the features of the methodand other configurations of the invention relating to killing firstbacteria instead relate mutatis mutandis to killing first archaea.

In an embodiment, the method comprises carrying out the method oftreating an acute microbial infection as described herein, and thusfeatures of that method as described herein are combinable with thepresent method of treating a pathogenic bacterial infection (ie, wherethe pathogenic bacterial infection is the acute microbial infection inthe first method). In an embodiment, the method comprises carrying outthe method of durably treating a microbial infection as describedherein, and thus features of that method as described herein arecombinable with the present method of treating a pathogenic bacterialinfection (ie, where the pathogenic bacterial infection is the microbialinfection in the first method). Any of the optional features of thefirst method herein may apply mutatis mutandis to the present method oftreating a pathogenic bacterial infection.

Aspects:—

Thus, the invention provides the following Aspects, which are optionalfeatures of Clauses above:—

-   -   1. The method of any one of Clauses 1-23, wherein the infection        is reduced at least 100-fold by the first 30 minutes of carrying        out step (b). Optionally, the infection is reduced at least        1000-fold by the first 30 minutes of carrying out step (b).        Optionally, the reduction in infection persists for 30 minutes        immediately after the first 30 minutes of carrying out step (b).        For example, the reduction can be assessed by determining the        difference in the number of bacteria of the first species or        strain in (i) a sample taken from the subject (eg, a blood        sample) immediately before commencement of the method and (ii) a        sample (of the same type as the sample of (i), eg, a blood        sample) taken from the subject at 30 minutes of the treatment.        For example, the samples may be assessed for the difference in        colony forming units (CFU)/ml sample, eg, when the samples have        been plated on agar in respective petri dishes and incubated        under identical conditions. Another example may use microscopic        counting of bacteria in samples, or other routine methods know        to the skilled addressee.    -   2. The method of any one of Clauses 1-23, wherein blood        infection of the subject by the first bacteria is reduced at        least 100- or 1000-fold by the first 30 minutes of carrying out        step (b).    -   3. The method of Aspect 2, wherein the blood is infected with        from 10⁵ to 10¹² (eg, 10⁷ to 10¹²) CFU/ml of the first bacteria        immediately before the treatment.    -   4. The method of any one of Clauses 1-23 or any preceding        Aspect, wherein the method comprises administering to the        subject a nucleic acid (eg, a RNA) and nuclease, wherein the        nucleic acid complexes with the nuclease to program the nuclease        to cut the target site in the first bacteria comprised by the        subject.    -   5. The method of Aspect 4, wherein the nuclease is administered        simultaneously or sequentially with the nucleic acid to the        subject.    -   6. The method of Aspect 4, wherein the subject comprises the        nuclease prior to administration of the nucleic acid to the        subject.    -   7. The method of any one of Aspects 4 to 6, wherein a plurality        phage are administered to the subject, wherein each phage        comprises a copy of the nucleic acid, wherein the phage infect        first bacteria comprised by the subject to deliver thereto the        nucleic acid.    -   8. The method of Aspect 7, wherein the ratio of administered        phage:first bacteria comprised by the subject is from 10 to 150.        For example, the ratio is from 10 to 100, ie, a multiplicity of        infection (MOI) of from 10 to 100.

The ratio can be determined, for example, using a sample (eg, a blood orgut sample) from a human or animal subject immediately before thetreatment and determining the number of bacteria per ml of blood or gutsample. The amount of phage to be administered can then be worked outaccording to the determination using the sample.

-   -   9. The method of any one of Clauses 1-23 or any preceding        Aspect, wherein the infection is an infection of the lungs,        brain, skin, abdomen or urinary tract.    -   10. The method of any one of Clauses 1-23 or any preceding        Aspect, wherein the subject has undergone surgery, is on an        immunosuppressant medication, suffering from burns, suffering        from diabetes, suffering from cancer or is suffering from a        chronic disease.    -   11. The method of any one of Clauses 1-23 or any preceding        Aspect, wherein the subject is a human over 65 years of age or        is a paediatric patient.    -   12. The method of any one of Clauses 1-23 or any preceding        Aspect, wherein the method treats or prevents sepsis in the        subject.    -   13. The method of Clause 12, wherein at the start of the        treatment, the subject (eg, a human) has a temperature of        <36° C. or >38° C.; a heart rate of >90/min, a respiratory rate        of >20 breaths/min or PaCO₂<4.3 kPa; and white blood cell count        of <4000/mm³ or >12,000/mm³.    -   14. The method of Clause 12 or 13, wherein at the start of the        treatment, the subject (eg, a human) has presence of two or more        of the following: abnormal body temperature, abnormal heart        rate, abnormal respiratory rate, abnormal blood gas and abnormal        white blood cell count.        Immune Checkpoint Modulation

Immune checkpoints of the invention either turn up a signal (e.g.,co-stimulatory molecules) or turn down a signal. Inhibitory immunecheckpoint molecules that may be targeted by immune checkpointmodulation in the invention include adenosine A2A receptor (AZAR), B7-H3(also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxicT-lymphocyte-associated protein 4 (CTLA-4, also known as CD 152),indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR),lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cellimmunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Igsuppressor of T cell activation (VISTA). In particular, the immunecheckpoint inhibitors target the PD-1 axis and/or CTLA-4.

The immune checkpoint inhibitors may be drugs such as small molecules,recombinant forms of ligand or receptors, or, antibodies, such as humanantibodies (e.g., WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64,2012; both incorporated herein by reference). Known inhibitors of theimmune checkpoint proteins or analogs thereof may be used, in particularchimerised, humanised or human forms of antibodies may be used. As theskilled person will know, alternative and/or equivalent names may be inuse for certain antibodies mentioned in the present disclosure. Suchalternative and/or equivalent names are interchangeable in the contextof the present invention. For example it is known that lambrolizumab isalso known under the alternative and equivalent names MK-3475 andpembrolizumab.

It is contemplated that any of the immune checkpoint inhibitors that areknown in the art to stimulate immune responses may be used. Thisincludes inhibitors that directly or indirectly stimulate or enhanceantigen-specific T-lymphocytes. These immune checkpoint inhibitorsinclude, without limitation, agents targeting immune checkpoint proteinsand pathways involving PD-L2, LAG3, BTLA, B7H4 and TIM3. For example,LAG3 inhibitors known in the art include soluble LAG3 (IMP321, orLAG3-Ig disclosed in WO2009044273) as well as mouse or humanizedantibodies blocking human LAG3 (e.g., IMP701 disclosed in WO2008132601),or fully human antibodies blocking human LAG3 (such as disclosed in EP2320940). Another example is provided by the use of blocking agentstowards BTLA, including without limitation antibodies blocking humanBTLA interaction with its ligand (such as 4C7 disclosed inWO2011014438). Yet another example is provided by the use of agentsneutralizing B7H4 including without limitation antibodies to human B7H4(disclosed in WO 2013025779, and in WO2013067492) or soluble recombinantforms of B7H4 (such as disclosed in US20120177645). Yet another exampleis provided by agents neutralizing B7-H3, including without limitationantibodies neutralizing human B7-H3 (e.g. MGA271 disclosed as BRCA84Dand derivatives in US 20120294796). Yet another example is provided byagents targeting TIM3, including without limitation antibodies targetinghuman TIM3 (e.g. as disclosed in WO 2013006490 A2 or the anti-humanTIM3, blocking antibody F38-2E2 disclosed by Jones et ah, J Exp Med.2008; 205(12):2763-79).

A. PD-1 Axis Antagonists

T cell dysfunction or anergy occurs concurrently with an induced andsustained expression of the inhibitory receptor, programmed death 1polypeptide (PD-1). Thus, therapeutic targeting of PD-1 and othermolecules which signal through interactions with PD-1, such asprogrammed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2)is provided herein. PD-L1 is overexpressed in many cancers and is oftenassociated with poor prognosis (Okazaki T et ah, Intern. Immun 200719(7):813). Thus, improved methods of treating cancer by inhibiting thePD-L1/PD-1 interaction in combination with modulating the microbiome isprovided herein.

For example, PD-1 axis binding antagonists include a PD-1 bindingantagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.Alternative names for “PD-1” include CD279 and SLEB2. Alternative namesfor “PD-L1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for“PD-L2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1,PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect, the PD-1 ligand binding partners are PD-L1 and/orPD-L2. In another embodiment, a PD-L1 binding antagonist is a moleculethat inhibits the binding of PD-L1 to its binding partners. In aspecific aspect, PD-L1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PD-L2 binding antagonist is a molecule that inhibits thebinding of PD-L2 to its binding partners. In a specific aspect, a PD-L2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide. Exemplary antibodies are described in U.S. Pat. Nos.8,735,553, 8,354,509, and 8,008,449, all incorporated herein byreference. Other PD-1 axis antagonists for use in the methods providedherein are known in the art such as described in U.S. Patent ApplicationNo. US20140294898, US2014022021, and US20110008369, all incorporatedherein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1antibody {e.g., a human antibody, a humanized antibody, or a chimericantibody). In some embodiments, the anti-PD-1 antibody is selected fromthe group consisting of nivolumab, pembrolizumab, and CT-011. In someembodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPDL1 or PD-L2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence). In some embodiments, the PD-1 bindingantagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106,ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described inWO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475,lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibodydescribed in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is ananti-PD-1 antibody described in WO2009/101611. AMP-224, also known asB7-DCIg, is a PD-L2-Fc fusion soluble receptor described inWO2010/027827 and WO2011/066342. Additional PD-1 binding antagonistsinclude pidilizumab, also known as CT-011, MEDI0680, also known asAMP-514, and REGN2810.

In some embodiments, the immune checkpoint inhibitor is a PD-L1antagonist such as durvalumab, also known as MEDI4736, atezolizumab,also known as MPDL3280A, or avelumab, also known as MSB00010118C. Incertain aspects, the immune checkpoint inhibitor is a PD-L2 antagonistsuch as rHIgM12B7. In some aspects, the immune checkpoint inhibitor is aLAG-3 antagonist such as, but not limited to, IMP321, and BMS-986016.The immune checkpoint inhibitor may be an adenosine Ata receptor (A2aR)antagonist such as PBF-509.

In some embodiments, any antibody described herein (such as an anti-PD-1antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody) furthercomprises a human or murine constant region. In a still further aspect,the human constant region is selected from the group consisting of IgG1,IgG2, IgG2, IgG3, and IgG4. In a still further specific aspect, thehuman constant region is IgG1. In a still further aspect, the murineconstant region is selected from the group consisting of IgG1, IgG2A,IgG2B, and IgG3. In a still further specific aspect, the antibody hasreduced or minimal effector function. In a still further specificaspect, the minimal effector function results from production inprokaryotic cells. In a still further specific aspect the minimaleffector function results from an “effector-less Fc mutation” oraglycosylation. Glycosylation of antibodies is typically either N-linkedor O-linked. N-linked refers to the attachment of the carbohydratemoiety to the side chain of an asparagine residue. The tripeptidesequences asparagine-X-serine and asparagine-X-threonine, where X is anyamino acid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxy amino acid, mostcommonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites form an antibodyis conveniently accomplished by altering the amino acid sequence suchthat one of the above-described tripeptide sequences (for N-linkedglycosylation sites) is removed. The alteration may be made bysubstitution of an asparagine, serine or threonine residue within theglycosylation site another amino acid residue (e.g., glycine, alanine ora conservative substitution).

The antibody or antigen binding fragment thereof, may be made usingmethods known in the art, for example, by a process comprising culturinga host cell containing nucleic acid encoding any of the previouslydescribed anti-PD-L1, anti-PD-1, or anti-PD-L2 antibodies orantigen-binding fragment in a form suitable for expression, underconditions suitable to produce such antibody or fragment, and recoveringthe antibody or fragment.

B. CTLA-4

Another immune checkpoint that can be targeted in the methods providedherein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), alsoknown as CD152. The complete cDNA sequence of human CTLA-4 has theGenbank accession number L15006. CTLA-4 is found on the surface of Tcells and acts as an “off switch when bound to CD80 or CD86 on thesurface of antigen-presenting cells. CTLA4 is a member of theimmunoglobulin superfamily that is expressed on the surface of Helper Tcells and transmits an inhibitory signal to T cells. CTLA4 is similar tothe T-cell co-stimulatory protein, CD28, and both molecules bind to CD80and CD86, also called B7-1 and B7-2 respectively, on antigen-presentingcells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28transmits a stimulatory signal. Intracellular CTLA4 is also found inregulatory T cells and may be important to their function. T cellactivation through the T cell receptor and CD28 leads to increasedexpression of CTLA-4, an inhibitory receptor for B7 molecules.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody), an antigen binding fragment thereof, an immunoadhesin, afusion protein, or oligopeptide.

Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom)suitable for use in the present methods can be generated using methodswell known in the art. Alternatively, art recognized anti-CTLA-4antibodies can be used. For example, the anti-CTLA-4 antibodiesdisclosed in: U.S. Pat. No. 8,119,129, WO 01/14424, WO 98/42752; WO00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab),U.S. Pat. No. 6,207,156; Hurwitz et al, 1998; can be used in the methodsdisclosed herein. The teachings of each of the aforementionedpublications are hereby incorporated by reference. Antibodies thatcompete with any of these art-recognized antibodies for binding toCTLA-4 also can be used. For example, a humanized CTLA-4 antibody isdescribed in International Patent Application No. WO2001014424,WO2000037504, and U.S. Pat. No. 8,017,114; all incorporated herein byreference.

An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1,MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variantsthereof (see, e.g., WO01/14424). In other embodiments, the antibodycomprises the heavy and light chain CDRs or VRs of ipilimumab.Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2,and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 andCDR3 domains of the VL region of ipilimumab. In another embodiment, theantibody competes for binding with and/or binds to the same epitope onCTLA-4 as the above-mentioned antibodies. In another embodiment, theantibody has at least about 90% variable region amino acid sequenceidentity with the above-mentioned antibodies (e.g., at least about 90%,95%, or 99% variable region identity with ipilimumab).

Other molecules for modulating CTLA-4 include soluble CTLA-4 ligands andreceptors such as described in U.S. Pat. Nos. 5,844,905, 5,885,796 andInternational Patent Application Nos. WO1995001994 and WO1998042752; allincorporated herein by reference, and immunoadhesins such as describedin U.S. Pat. No. 8,329,867, incorporated herein by reference.

C. Killer Immunoglobulin-Like Receptor (KIR)

Another immune checkpoint inhibitor for use in the present invention isan anti-KIR antibody. Anti-human-KIR antibodies (or VH/VL domainsderived therefrom) suitable for use in the present methods can begenerated using methods well known in the art.

Alternatively, art recognized anti-KIR antibodies can be used. Theanti-KIR antibody can be cross-reactive with multiple inhibitory KIRreceptors and potentiates the cytotoxicity of NK cells bearing one ormore of these receptors. For example, the anti-KIR antibody may bind toeach of KIR2D2DL1, KIR2DL2, and KIR2DL3, and potentiate NK cell activityby reducing, neutralizing and/or reversing inhibition of NK cellcytotoxicity mediated by any or all of these KIRs. In some aspects, theanti-KIR antibody does not bind KIR2DS4 and/or KIR2DS3. For example,monoclonal antibodies 1-7F9 (also known as IPH2101), 14F1, 1-6F1 and1-6F5, described in WO 2006/003179, the teachings of which are herebyincorporated by reference, can be used. Antibodies that compete with anyof these art-recognized antibodies for binding to KIR also can be used.Additional art-recognized anti-KIR antibodies which can be used include,for example, those disclosed in WO 2005/003168, WO 2005/009465, WO2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO2010/065939, WO 2012/071411 and WO 2012/160448.

An exemplary anti-KIR antibody is lirilumab (also referred to asBMS-986015 or IPH2102). In other embodiments, the anti-KIR antibodycomprises the heavy and light chain complementarity determining regions(CDRs) or variable regions (VRs) of lirilumab. Accordingly, in oneembodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains ofthe heavy chain variable (VH) region of lirilumab, and the CDR1, CDR2and CDR3 domains of the light chain variable (VL) region of lirilumab.In another embodiment, the antibody has at least about 90% variableregion amino acid sequence identity with lirilumab.

Examples of cancers contemplated for treatment include lung cancer, headand neck cancer, breast cancer, pancreatic cancer, prostate cancer,renal cancer, bone cancer, testicular cancer, cervical cancer,gastrointestinal cancer, lymphomas, pre-neoplastic lesions in the lung,colon cancer, melanoma, metastatic melanoma, basal-cell skin cancer,squamous-cell skin cancer, dermatofibrosarcoma protuberans, Merkel cellcarcinoma, Kaposi's sarcoma, keratoacanthoma, spindle cell tumours,sebaceous carcinomas, microcystic adnexal carcinoma, Paget's disease ofthe breast, atypical fibroxanthoma, leiomyosarcoma, and angiosarcoma,Lentigo Maligna, Lentigo Maligna Melanoma, Superficial SpreadingMelanoma, Nodular Melanoma, Acral Lentiginous Melanoma, DesmoplasticMelanoma, and bladder cancer.

In some embodiments, the subject has cancer that is resistant (has beendemonstrated to be resistant) to one or more anti-cancer therapies. Insome embodiments, resistance to anti-cancer therapy includes recurrenceof cancer or refractory cancer. Recurrence may refer to the reappearanceof cancer, in the original site or a new site, after treatment. In someembodiments, resistance to anti-cancer therapy includes progression ofthe cancer during treatment with the anti-cancer therapy. In someembodiments, the cancer is at early stage or at late stage. The subjectmay have a cancer that expresses (has been shown to express e.g., in adiagnostic test) PD-L1 biomarker. In some embodiments, the patient'scancer expresses low PD-L1 biomarker. In some embodiments, the patient'scancer expresses high PD-L1 biomarker. The PD-L1 biomarker can bedetected in the sample using a method selected from the group consistingof FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry,immunofluorescence, radioimmunoassay, dot blotting, immunodetectionmethods, HPLC, surface plasmon resonance, optical spectroscopy, massspectrometery, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq,microarray analysis, SAGE, MassARRAY technique, and FISH, andcombinations thereof.

In some embodiments, the cancer has low levels of T cell infiltration.In some embodiments, the cancer has no detectable T cell infiltrate. Insome embodiments, the cancer is a non-immunogenic cancer (e.g.,non-immunogenic colorectal cancer and/or ovarian cancer).

For example, a therapeutically effective or sufficient amount of theimmune checkpoint inhibitor, such as an antibody, is administered to ahuman will be in the range of about 0.01 to about 50 mg/kg of patientbody weight whether by one or more administrations. In some embodiments,the antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg,about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example.In some embodiments, the antibody is administered at 15 mg/kg. However,other dosage regimens may be useful. In one embodiment, an anti-PD-L1antibody described herein is administered to a human at a dose of about100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of21-day cycles. The dose may be administered as a single dose or asmultiple doses (e.g., 2 or 3 doses), such as infusions. The progress ofthis therapy is easily monitored by conventional techniques.

Anti-Cancer and Other Therapies

In some embodiments, the immune checkpoint inhibitor may be administeredin combination with at least one additional therapeutic. The additionaltherapy may be a cancer therapy such as radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy.

In an example, the therapy of the cancer (whether with or withoutadministration of an immune checkpoint inhibitor) or any other disease(eg, viral infection or autoimmune disease) may radiation therapy,surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNAtherapy, immunotherapy, bone marrow transplantation, nanotherapy ormonoclonal antibody therapy. The therapy may be a combination of theforegoing. An additional therapy may be administered

In some embodiments, the therapy (or the additional cancer therapy) isthe administration of a small molecule enzymatic inhibitor oranti-metastatic agent. In some embodiments, the additional therapy isthe administration of side-effect limiting agents (e.g., agents intendedto lessen the occurrence and/or severity of side effects of treatment,such as anti-nausea agents, etc.).

In some embodiments, the therapy (or the additional cancer therapy) isradiation therapy. In some embodiments, the therapy (or the additionalcancer therapy) is surgery. In some embodiments, the therapy (or theadditional cancer therapy) is a combination of radiation therapy andsurgery. In some embodiments, the therapy (or the additional cancertherapy) is gamma irradiation. In some embodiments, the therapy (or theadditional cancer therapy) is therapy targeting PBK/AKT/mTOR pathway,HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/orchemopreventative agent. The therapy (or the additional cancer therapy)may be one or more of the chemotherapeutic agents known in the art.

Administration of any compound or therapy of the present embodiments toa patient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the agents.Therefore, in some embodiments there is a step of monitoring toxicitythat is attributable to combination therapy.

The therapy can comprise or consist of administration to the subject ofany of the following:—

1. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present embodiments. The term “chemotherapy” refers to the use ofdrugs to treat cancer. A “chemotherapeutic agent” is used to connote acompound or composition that is administered in the treatment of cancer.These agents or drugs are categorized by their mode of activity within acell, for example, whether and at what stage they affect the cell cycle.Alternatively, an agent may be characterized based on its ability todirectly cross-link DNA, to intercalate into DNA, or to inducechromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such asthiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines, includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065 (eg, itsadozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (eg, the synthetic analogues, KW-2189 and CB 1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards, such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, and uracil mustard; nitrosureas, such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics, such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (eg,morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolicacid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; anti-metabolites, such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogues, such asdenopterin, pteropterin, and trimetrexate; purine analogs, such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs, such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens, such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals, such as mitotane andtrilostane; folic acid replenisher, such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharidecomplex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (eg, T-2toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g.,paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan(e.g., CPT-11); topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids, such as retinoic acid;capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien,navelbine, farnesyl-protein tansferase inhibitors, transplatinum, andpharmaceutically acceptable salts, acids, or derivatives of any of theabove{circumflex over ( )}

2. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumour cells. Other forms of DNA damagingfactors are also contemplated, such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), andUV-irradiation. It is most likely that all of these factors affect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 weeks), to singledoses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes varywidely, and depend on the half-life of the isotope, the strength andtype of radiation emitted, and the uptake by the neoplastic cells.

3. Immunotherapy

The skilled artisan will understand that immunotherapies may be used incombination or in conjunction with the methods described herein. In thecontext of cancer treatment, immunotherapeutics generally rely on theuse of immune effector cells and molecules to target and destroy cancercells. Rituximab (RITUXAN®) is an example of an immunotherapy. Theimmune effector may be, for example, an antibody specific for a markeron the surface of a tumour cell. The antibody alone may serve as aneffector of therapy or it may recruit other cells to actually effectcell killing. The antibody also may be conjugated to a drug or toxin(chemo therapeutic, radionuclide, ricin A chain, cholera toxin,pertussis toxin, etc.) and serve as a targeting agent. Alternatively,the effector may be a lymphocyte carrying a surface molecule thatinteracts, either directly or indirectly, with a tumour cell target.Various effector cells include cytotoxic T cells and NK cells.

In an example, the immunotherapy comprises adoptive cell therapy, suchas CAR-T administration, eg, anti-CD19 or CD20 CAR-T administration.

In an example, the immunotherapy comprises or consists of administrationof an IL-2 (eg, a truncated IL-2 or pegylated IL-2 or Fc-fused IL-2).

Antibody-drug conjugates have emerged as a breakthrough approach to thedevelopment of cancer therapeutics. Antibody-drug conjugates (ADCs)comprise monoclonal antibodies (MAbs) that are covalently linked tocell-killing drugs. This approach combines the high specificity of MAbsagainst their antigen targets with highly potent cytotoxic drugs,resulting in “armed” MAbs that deliver the payload (drug) to tumourcells with enriched levels of the antigen. Targeted delivery of the drugalso minimizes its exposure in normal tissues, resulting in decreasedtoxicity and improved therapeutic index. The approval of two ADC drugs,ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumabemtansine or T-DM1) in 2013 by FDA validated the approach. There arecurrently more than 30 ADC drug candidates in various stages of clinicaltrials for cancer treatment. As antibody engineering and linker-payloadoptimization are becoming more and more mature, the discovery anddevelopment of new ADCs are increasingly dependent on the identificationand validation of new targets that are suitable to this approach and thegeneration of targeting MAbs. Two criteria for ADC targets areupregulated/high levels of expression in tumour cells and robustinternalization.

In one aspect of immunotherapy, the tumour cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumour markers exist and any of these may be suitablefor targeting in the context of the present embodiments. Common tumourmarkers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68,TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor,erb B, and pi 55. An alternative aspect of immunotherapy is to combineanticancer effects with immune stimulatory effects. Immune stimulatingmolecules also exist including: cytokines, such as IL-2, IL-4, IL-12,GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growthfactors, such as FLT3 ligand.

4. Surgery

The cancer or other disease or condition may be treated by surgery inthe invention.

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative, andpalliative surgery. Curative surgery includes resection in which all orpart of cancerous tissue is physically removed, excised, and/ordestroyed and may be used in conjunction with other therapies, such asthe treatment of the present embodiments, chemotherapy, radiotherapy,hormonal therapy, gene therapy, immunotherapy, and/or alternativetherapies. Tumour resection refers to physical removal of at least partof a tumour. In addition to tumour resection, treatment by surgeryincludes laser surgery, cryosurgery, electro surgery, andmicroscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumour, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection, or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

5. Bacterial Transplants

In an embodiment, the therapy comprises administering to the subject abacterial transplant, eg, a faecal microbial transplant, comprisingdefined bacteria. For example, the transplant is any compositiondisclosed in WO2018064165, the disclosure of which (especially thecompositions therein) are incorporated herein by reference in itsentirety for possible application in the present invention. For example,the transplant is according to any of the following Paragraphs (tablesnd sequence numbers referring to the tables and sequences inWO2018064165, which are explicitly incorporated herein for possible usein the Claims):—

-   -   1. A composition comprising at least one isolated or purified        population of bacteria belonging to one or more of the families        Ruminococcaceae, Clostridiaceae, Lachnospiraceae,        Micrococcaceae, and/or Veilonellaceae.    -   2. A composition comprising at least two isolated or purified        populations of bacteria belonging to one or more of the families        Ruminococcaceae, Clostridiaceae, Lachnospiraceae,        Micrococcaceae, and/or Veilonellaceae.    -   3. The composition of Paragraph 1 or Paragraph 2, wherein each        of the populations of bacteria is present in the composition at        a concentration of at least 10{circumflex over ( )}3 CFU.    -   4. The composition of Paragraph 1 or Paragraph 2, wherein the        composition is a live bacterial product or a live biotherapeutic        product.    -   5. The composition of Paragraph 1 or Paragraph 2, wherein the at        least one isolated or purified population bacteria or the at        least two isolated or purified populations of bacteria are        provided as bacterial spores.    -   6. The composition of Paragraph 1 or Paragraph 2, wherein the at        least one population of bacteria or the at least two isolated or        purified populations of bacteria belong to Clostridiales Family        XII and/or Clostridiales Family XIII.    -   7. The composition of Paragraph 1 or Paragraph 2, wherein the at        least one isolated or purified population bacteria or the at        least two isolated or purified populations of bacteria belong to        the family Ruminococcaceae and/or of the family Clostridiaceae.    -   8. The composition of Paragraph 1 or Paragraph 2, wherein the        population of bacteria belonging to the family Ruminococcaceae        is further defined as a population of bacteria belonging to the        genus Ruminococcus.    -   9. The composition of Paragraph 8, wherein the population of        bacteria belonging to the genus Ruminococcus is further defined        as a population of bacteria belonging to the species        Ruminococcus bromii.    -   10. The composition of Paragraph 1 or Paragraph 2, wherein the        population of bacteria belonging to the family Ruminococcaceae        is further defined as a population of bacteria belonging to the        genus Faecalibacterium.    -   11. The composition of Paragraph 10, wherein the population of        bacteria belonging to the genus Faecalibacterium is further        defined as a population of bacteria belonging to the species        Faecalibacterium prausnitzii.    -   12. The composition of Paragraph 1 or Paragraph 2, wherein the        population of bacteria belonging to the family Micrococcaceae is        further defined as a population of bacteria belonging to the        genus Rothia.    -   13. The composition of Paragraph 1 or Paragraph 2, wherein the        composition further comprises a population of bacteria belonging        to the species Porphyromonas pasteri, the species Clostridium        hungatei, the species Phascolarctobacterium faecium, the genus        Peptoniphilus, and/or the class Mollicutes.    -   14. The composition of Paragraph 1 or Paragraph 2, wherein the        composition is essentially free of populations of bacteria        belonging to the order Bacteroidales.    -   15. The composition of Paragraph 1 or Paragraph 2, wherein the        at least one isolated or purified population bacteria or the at        least two isolated or purified populations of bacteria belongs        to one or more of the species, subspecies or bacterial strains        selected from the group consisting of the species in Table 1        with an enrichment index (ei) greater than 0.5.    -   16. The composition of Paragraph 1 or Paragraph 2, wherein the        at least one isolated or purified population bacteria or the at        least two isolated or purified populations of bacteria are        selected from the group consisting of the species in Table 1        with an “ei” equal to 1.    -   17. The composition of Paragraph 1 or Paragraph 2, wherein the        at least one isolated or purified population bacteria or the at        least two isolated or purified populations of bacteria comprise        a 16S ribosomal RNA (rRNA) nucleotide sequence that is at least        90% identical (eg, at least 91, 92, 93, 94, 95, 96, 97, 98 or        99% identical) to the 16S rRNA nucleotide sequence of bacteria        identified by NCBI Taxonomy IDs selected from the group        consisting of NCBI Taxonomy ID: 717959, 587, 758823, 649756,        44749, 671218, 1264, 1122135, 853, 484018, 46503, 54565, 290052,        216931, 575978, 433321, 1796646, 213810, 228924, 290054, 1509,        1462919, 29375, 337097, 1298596, 487174, 642492, 1735, 1297424,        742766, 46680, 132925, 411467, 1318465, 1852367, 1841857,        169679, 1175296, 259063, 172901, 39488, 57172, 28118, 166486,        28133, 1529, 694434, 1007096, 84030, 56774, 102148, 626947,        216933, 1348613, 1472417, 100176, 824, 1471761, 1297617, 288966,        1317125, 28197, 358743, 264639, 1265, 1335, 66219, 69473,        115117, 341220, 1732, 873513, 396504, 1796619, 45851, 2741,        105841, 86332, 1349822, 84037, 180311, 54291, 1217282, 762984,        1185412, 154046, 663278, 1543, 398512, 69825, 1841867, 1535,        1510, 84026, 1502, 1619234, 39497, 1544, 29343, 649762, 332095,        536633, 1033731, 574930, 742818, 177412, 1121308, 419208,        1673717, 55779, 28117, 626937, 180332, 1776382, 40519, 34062,        40518, 74426, 1216062, 293826, 850, 645466, 474960, 36835,        115544, 1515, 88431, 216932, 1417852, 39492, 1583, 420247,        118967, 169435, 37658, 138595, 31971, 100886, 1197717, 234908,        537007, 319644, 168384, 915173, 95159, 1816678, 626940, 501571,        1796620, 888727, 1147123, 376806, 1274356, 1267, 39495, 404403,        1348, 253314, 258515, 33033, 1118061, 357276, 214851, 320502,        217731, 246787, 29371, 649764, 901, 29374, 33043, 39778, 682400,        871665, 160404, 745368, 408, 1584, 333367, 47246, 1096246,        53342, 438033, 351091, 1796622, 1776384, 817, 48256, 720554,        500632, 36849, 301302, 879970, 655811, 264463, 1532, 285, 995,        242750, 29539, 1432052, 622312, 1796636, 1337051, 328814, 28446,        1492, 820, 39496, 52786, 1549, 1796618, 582, 46507, 109327,        1531, 1382, 33039, 311460, 230143, 216935, 539, 35519, 1681,        328813, 214853, 89014, 1121115, 1585974, 29466, 1363, 292800,        270498, 214856, 142877, 133926, 209880, 179628, 1121102, 105612,        1796615, 39777, 29353, 1579, 163665, 53443, 261299, 1302,        1150298, 938289, 358742, 471875, 938278, 1796613, 1118057,        1077144, 1737, 218205, 1121298, 684066, 433659, 52699, 204516,        706562, 253257, 328812, 1280, 147802, 58134, 1335613, 891,        585394, 1582, 235931, 308994, 1589, 1682, 1736, 28129, 178001,        551788, 2051, 856, 118562, 101070, 515619, 40215, 187979, 82979,        29363, 1776391, 1285191, 84112, 157688, 38304, 36850, 341694,        287, 75612, 818, 371674, 338188, 88164, 588581, 676965, 546271,        1236512, 178338, 862517, 157687, 158, 51048, 1583331, 529,        888745, 394340, 40545, 855, 553973, 938293, 93063, 708634,        179995, 1351, 476652, 1464038, 555088, 237576, 879566, 1852371,        742727, 1377, 35830, 997353, 218538, 83771, 1605, 28111, 131109,        46609, 690567, 46206, 155615, 51616, 40542, 203, 294, 1034346,        156456, 80866, 554406, 796942, 1002367, 29347, 796944, 61592,        487175, 1050201, 762948, 137732, 1211819, 1019, 272548, 1717,        384636, 216940, 2087, 45634, 466107, 1689, 47678, 575, 979627,        840, 1660, 1236517, 617123, 546, 28135, 82171, 483, 501496,        99656, 1379, 84032, 39483, 1107316, 584, 28124, 1033744, 657309,        536441, 76123, 1118060, 89152, 76122, 303, 1541, 507751, 515620,        38302, 53419, 726, 40324, 1796610, 988946, 1852370, 1017,        1168289, 76936, 94869, 1161098, 215580, 1125779, 327575, 549,        1450648 and 478.    -   18. The composition of Paragraph 1 or Paragraph 2, wherein the        at least one isolated or purified population of bacteria or the        at least two isolated or purified populations of bacteria are a        species, subspecies or bacterial strains comprising a 16S rRNA        gene sequence at least 80% identical (eg, at least 85, 90, 95 or        98% identical) to any one of the sequences of SEQ ID NOs: 1-876        in WO2018064165.    -   19. The composition of Paragraph 1 or Paragraph 2, wherein the        at least one isolated or purified population bacteria or the at        least two isolated or purified populations of bacteria belong to        the species, subspecies or bacterial strains selected from the        group consisting of Bacteroides coagulans, Clostridium        aldenense, Clostridium aldrichii, Clostridium alkalicellulosi,        Clostridium amygdalinum, Clostridium asparagiforme, Clostridium        cellulosi, Clostridium citroniae, Clostridium clariflavum DSM        19732, Clostridium clostridioforne, Clostridium colinum,        Clostridium fimetarium, Clostridium hiranonis, Clostridium        hungatei, Clostridium hylemonae DSM 15053, Clostridium indolis,        Clostridium lactatifermentans, Clostridium leptum, Clostridium        methylpentosum, Clostridium oroticum, Clostridium papyrosolvens        DSM 2782, Clostridium populeti, Clostridium propionicum,        Clostridium saccharolyticum, Clostridium scindens, Clostridium        sporosphaeroides, Clostridium stercorarium, Clostridium        straminisolvens, Clostridium sufflavum, Clostridium termitidis,        Clostridium thermosuccino genes, Clostridium viride, Clostridium        xylanolyticum, Desulfotomaculum guttoideum, Eubacterium rectale        ATCC 33656, Eubacterium dolichum, Eubacterium eligens ATCC        27750, Eubacterium hallii, Eubacterium infirmum, Eubacterium        siraeum, Eubacterium tenue, Ruminococcus torques,        Acetanaerobacterium elongatum, Acetatifactor muris, Acetivibrio        cellulolyticus, Acetivibrio ethanolgignens, Acholeplasma        brassicae 0502, Acholeplasma parvum, Acholeplasma vituli,        Acinetobacter junii, Actinobacillus porcinus, Actinomyces        bowdenii, Actinomyces dentalis, Actinomyces odontolyticus,        Acutalibacter muris, Aerococcus viridans, Aeromicrobium        fastidiosum, Alistipes finegoldii, Alistipes obesi, Alistipes        onderdonkii, Alistipes putredinis, Alistipes shahii, Alistipes        shahii WAL 8301, Alistipes timonensis JC136, Alkalibacter        saccharofermentans, Alkaliphilus metalliredigens QYMF,        Allisonella histaminiformans, Allobaculum stercoricanis DSM        13633, Alloprevotella rava, Alloprevotella tannerae,        Anaerobacterium chartisolvens, Anaerobiospirillum thomasii,        Anaerobium acetethylicum, Anaerococcus octavius NCTC 9810,        Anaerococcus provenciensis, Anaerococcus vaginalis ATCC 51170,        Anaerocolumna jejuensis, Anaerofilum agile, Anaerofustis        stercorihominis, Anaeroglobus geminatus, Anaeromassilibacillus        senegalensis, Anaeroplasma abactoclasticum, Anaerorhabdus        furcosa, Anaerosporobacter mobilis, Anaerostipes butyraticus,        Anaerostipes caccae, Anaerostipes hadrus, Anaerotruncus        colihominis, Anaerovorax odorimutans, Anoxybacillus rupiensis,        Aquabacterium limnoticum, Arcobacter butzleri, Arthrospira        platensis, Asaccharobacter celatus, Atopobium parvulum,        Bacteroides caccae, Bacteroides caecimuris, Bacteroides        cellulosilyticus, Bacteroides clarus YIT 12056, Bacteroides        dorei, Bacteroides eggerthii, Bacteroides finegoldii,        Bacteroides fragilis, Bacteroides gallinarum, Bacteroides        massiliensis, Bacteroides oleiciplenus YIT 12058, Bacteroides        plebeius DSM 17135, Bacteroides rodentium JCM 16496, Bacteroides        thetaiotaomicron, Bacteroides uniformis, Bacteroides        xylanisolvens XB1A, Bacteroides xylanolyticus, Barnesiella        intestinihominis, Beduini massiliensis, Bifidobacterium bifidum,        Bifidobacterium dentium, Bifidobacterium longum subsp. infantis,        Blautia caecimuris, Blautia coccoides, Blautia faecis, Blautia        glucerasea, Blautia hansenii DSM 20583, Blautia        hydrogenotrophica, Blautia luti, Blautia luti DSM 14534, Blautia        wexlerae DSM 19850, Budvicia aquatica, Butyricicoccus        pullicaecorum, Butyricimonas paravirosa, Butyrivibrio crossotus,        Caldicoprobacter oshimai, Caloramator coolhaasii, Caloramator        proteoclasticus, Caloramator quimbayensis, Campylobacter        gracilis, Campylobacter rectus, Campylobacter ureolyticus DSM        20703, Capnocytophaga gingivalis, Capnocytophaga leadbetteri,        Capnocytophaga sputigena, Casaltella massiliensis, Catabacter        hongkongensis, Catenibacterium mitsuokai, Christensenella        minuta, Christensenella timonensis, Chryseobacterium        taklimakanense, Citrobacter freundii, Cloacibacillus porcorum,        Clostridioides difficile ATCC 9689=DSM 1296, Clostridium        amylolyticum, Clostridium bowmanii, Clostridium butyricum,        Clostridium cadaveris, Clostridium colicanis, Clostridium        gasigenes, Clostridium lentocellum DSM 5427, Clostridium        oceanicum, Clostridium oryzae, Clostridium paraputrificum,        Clostridium pascui, Clostridium perfringens, Clostridium quinii,        Clostridium saccharobutylicum, Clostridium sporogenes,        Clostridium ventriculi, Collinsella aerofaciens, Comamonas        testosteroni, Coprobacter fastidiosus NSB1, Coprococcus        eutactus, Corynebacterium diphtheriae, Corynebacterium durum,        Corynebacterium mycetoides, Corynebacterium pyruviciproducens        ATCC BAA-1742, Corynebacterium tuberculostearicum, Culturomica        massiliensis, Cuneatibacter caecimuris, Defluviitalea        saccharophila, Delftia acidovorans, Desulfitobacterium        chlororespirans, Desulfitobacterium metallireducens,        Desulfosporosinus acididurans, Desulfotomaculum halophilum,        Desulfotomaculum intricatum, Desulfotomaculum tongense,        Desulfovibrio desulfuricans subsp. desulfuricans, Desulfovibrio        idahonensis, Desulfovibrio litoralis, Desulfovibrio piger,        Desulfovibrio simplex, Desulfovibrio zosterae, Desulfuromonas        acetoxidans, Dethiobacter alkaliphilus AHT 1,        Dethiosulfatibacter aminovorans, Dialister invisus, Dialister        propionicifaciens, Dielma fastidiosa, Dietzia alimentaria 72,        Dorea longicatena, Dysgonomonas gadei ATCC BAA-286, Dysgonomonas        mossii, Eggerthella lenta, Eikenella corrodens, Eisenbergiella        tayi, Emergencia timonensis, Enorma massiliensis phi,        Enterococcus faecalis, Enterorhabdus muris, Ethanoligenens        harbinense YUAN-3, Eubacterium coprostanoligenes, Eubacterium        limosum, Eubacterium oxidoreducens, Eubacterium sulci ATCC        35585, Eubacterium uniforme, Eubacterium ventriosum, Eubacterium        xylanophilum, Extibacter muris, Ezakiella peruensis,        Faecalibacterium prausnitzii, Faecalicoccus acidiformans,        Faecalitalea cylindroides, Filifactor villosus, Flavonifr actor        plautii, Flintibacter butyricus, Frisingicoccus caecimuris,        Fucophilus fucoidanolyticus, Fusicatenibacter saccharivorans,        Fusobacterium mortiferum, Fusobacterium nucleatum subsp.        vincentii, Fusobacterium simiae, Fusobacterium varium, Garciella        nitratireducens, Gemella haemolysans, Gemmiger Gordonibacter        urolithinfaciens, Gracilibacter thermotolerans JW/YJL-S1,        Granulicatella elegans, Guggenheimella bovis, Haemophilus        haemolyticus, Helicobacter typhlonius, Hespellia stercorisuis,        Holdemanella biformis, Holdemania massiliensis AP2, Howardella        ureilytica, Hungatella effluvii, Hungatella hathewayi,        Hydrogenoanaerobacterium saccharovorans, Ihubacter massiliensis,        Intestinibacter bartlettii, Intestinimonas butyriciproducens,        Irregularibacter muris, Kiloniella laminariae DSM 19542,        Kroppenstedtia guangzhouensis, Lachnoanaerobaculum orale,        Lachnoanaerobaculum umeaense, Lachnoclostridium phytofermentans,        Lactobacillus acidophilus, Lactobacillus algidus, Lactobacillus        animalis, Lactobacillus casei, Lactobacillus delbrueckii,        Lactobacillus formicalis, Lactobacillus iners, Lactobacillus        pentosus, Lactobacillus rogosae, Lactococcus garvieae,        Lactonifactor longoviformis, Leptotrichia buccalis, Leptotrichia        hofstadii, Leptotrichia hongkongensis, Leptotrichia wadei,        Leuconostoc inhae, Levyella massiliensis, Loriellopsis        cavernicola, Lutispora thermophila, Marinilabilia salmonicolor        JCM 21150, Marvinbryantia formatexigens, Mesoplasma photuris,        Methanobrevibacter smithii ATCC 35061, Methanomassiliicoccus        luminyensis BIO, Methylobacterium extorquens, Mitsuokella        jalaludinii, Mobilitalea sibirica, Mobiluncus curtisii,        Mogibacterium pumilum, Mogibacterium timidum, Moorella        glycerini, Moorella humiferrea, Moraxella nonliquefaciens,        Moraxella osloensis, Morganella morganii, Moryella indoligenes,        Muribaculum intestinale, Murimonas intestini, Natranaerovirga        pectinivora, Neglecta timonensis, Neisseria cinerea, Neisseria        oralis, Nocardioides mesophilus, Novibacillus thermophilus,        Ochrobactrum anthropi, Odoribacter splanchnicus, Olsenella        profusa, Olsenella uli, Oribacterium asaccharolyticum ACB7,        Oribacterium sinus, Oscillibacter ruminantium GHJ, Oscillibacter        valericigenes, Oxobacter pfennigii, Pantoea agglomerans,        Papillibacter cinnamivorans, Parabacteroides faecis,        Parabacteroides goldsteinii, Parabacteroides gordonii,        Parabacteroides merdae, Parasporobacterium paucivorans,        Parasutterella excrementihominis, Parasutterella secunda,        Parvimonas micra, Peptococcus niger, Peptoniphilus duerdenii        ATCC BAA-1640, Peptoniphilus grossensis ph5, Peptoniphilus        koenoeneniae, Peptoniphilus senegalensis JC140,        Peptostreptococcus stomatis, Phascolarctobacterium        succinatutens, Phocea massiliensis, Pontibacter indicus,        Porphyromonas bennonis, Porphyromonas endodontalis,        Porphyromonas pasteri, Prevotella bergensis, Prevotella buccae        ATCC 33574, Prevotella denticola, Prevotella enoeca, Prevotella        fusca JCM 17724, Prevotella loescheii, Prevotella nigrescens,        Prevotella oris, Prevotella pollens ATCC 700821, Prevotella        stercorea DSM 18206, Prev ote llamas silia timonensis,        Propionispira arcuata, Proteus mirabilis, Providencia rettgeri,        Pseudobacteroides cellulosolvens ATCC 35603=DSM 2933,        Pseudobutyrivibrio ruminis, Pseudoflavonifr actor capillosus        ATCC 29799, Pseudomonas aeruginosa, Pseudomonas fluorescens,        Pseudomonas mandelii, Pseudomonas nitroreducens, Pseudomonas        putida, Raoultella ornithinolytica, Raoultella planticola,        Raoultibacter massiliensis, Robinsoniella peoriensis, Romboutsia        timonensis, Roseburia faecis, Roseburia hominis A2-183,        Roseburia intestinalis, Roseburia inulinivorans DSM 16841,        Rothia dentocariosa ATCC 17931, Ruminiclostridium thermocellum,        Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus,        Ruminococcus champanellensis 18P13=JCM 17042, Ruminococcus        faecis JCM 15917, Ruminococcus flavefaciens, Ruminococcus        gauvreauii, Ruminococcus lactaris ATCC 29176, Rummeliibacillus        pycnus, Saccharofermentans acetigenes, Scardovia wiggsiae,        Schlegelella thermodepolymerans, Sedimentibacter hongkongensis,        Selenomonas sputigena ATCC 35185, Slackia exigua ATCC 700122,        Slackia piriformis YIT 12062, Solitalea canadensis,        Solobacterium moorei, Sphingomonas aquatilis, Spiroplasma        alleghenense, Spiroplasma chinense, Spiroplasma chrysopicola,        Spiroplasma culicicola, Spiroplasma lampyridicola, Sporobacter        termitidis, Staphylococcus aureus, Stenotrophomonas maltophilia,        Stomatobaculum longum, Streptococcus agalactiae ATCC 13813,        Streptococcus cristatus, Streptococcus equinus, Streptococcus        gordonii, Streptococcus lactarius, Streptococcus parauberis,        Subdoligranulum variabile, Succinivibrio dextrinosolvens,        Sutterella stercoricanis, Sutterella wadsworthensis,        Syntrophococcus sucromutans, Syntrophomonas zehnderi OL-4,        Terrisporobacter mayombei, Thermoleophilum album, Treponema        denticola, Treponema socranskii, Tyzzerella nexilis DSM 1787,        Vallitalea guaymasensis, Vallitalea pronyensis, Vampirovibrio        chlorellavorus, Veillonella atypica, Veillonella denticariosi,        Veillonella dispar, Veillonella parvula, Victivallis vadensis,        Vulcanibacillus modesticaldus and Weissella confusa.

In an example, the transplant comprises or consists of SER-109 orSER-262 (and optionally the condition is a C. dificile infection); VE202or SER-287 (and optionally the disease is ulcerative colitis); SER-301(and optionally the disease is IBD); SER-401 (and optionally thecondition is a cancer; eg, wherein the therapy further comprisesadministration of an anti-PD-1 axis antibody, eg, an anti-PD-1antibody); VE800 or SER-155 (and optionally the therapy furthercomprises the administration of a transplant, eg, a haematopoietic stemcell or solid organ transplant); EDP1066 or EDP1815 (and optionally thedisease is an inflammatory condition, eg, colitis, Crohn's disease,asthma, rheumatoid arthritis (RA), psoriasis, dermatitis (eg, atopicdermatitis) or IBD); or EDP1503 (and the disease is a cancer, eg,colorectal cancer, renal cell carcinoma, melanoma or a PD-1 relapsedcancer). In an example, the therapy comprises the administration ofSGM-1019, SG-2-0776 or EB8018 (and optionally the disease or conditionis NASH or IBD or an inflammatory condition, eg, colitis, Crohn'sdisease, asthma, rheumatoid arthritis (RA), psoriasis and dermatitis(eg, atopic dermatitis). Those starting “VE” are developed by VadantaBiosciences, SER are developed by Seres Therapeutics, EDP are developedby Evelo Biosciences, SG are developed by Second Genome and EB aredeveloped by Enterome.

In an example, the disease or condition herein is an inflammatorycondition, eg, colitis, Crohn's disease, asthma, rheumatoid arthritis(RA), psoriasis, dermatitis (eg, atopic dermatitis) or IBD.

6. Other Agents

It is contemplated that other agents may be used in combination withcertain aspects of the present embodiments to improve the therapeuticefficacy of treatment. These additional agents include agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Increases inintercellular signalling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighbouringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with certain aspectsof the present embodiments to improve the anti-hyperproliferativeefficacy of the treatments. Inhibitors of cell adhesion are contemplatedto improve the efficacy of the present embodiments. Examples of celladhesion inhibitors are focal adhesion kinase (FAKs) inhibitors andLovastatin. It is further contemplated that other agents that increasethe sensitivity of a hyperproliferative cell to apoptosis, such as theantibody c225, could be used in combination with certain aspects of thepresent embodiments to improve the treatment efficacy.

Diseases and Conditions

Optionally, the disease or condition is selected from

-   -   (a) A neurodegenerative disease or condition;    -   (b) A brain disease or condition;    -   (c) A CNS disease or condition;    -   (d) Memory loss or impairment;    -   (e) A heart or cardiovascular disease or condition, eg, heart        attack, stroke or atrial fibrillation;    -   (f) A liver disease or condition;    -   (g) A kidney disease or condition, eg, chronic kidney disease        (CKD);    -   (h) A pancreas disease or condition;    -   (i) A lung disease or condition, eg, cystic fibrosis or COPD;    -   (j) A gastrointestinal disease or condition;    -   (k) A throat or oral cavity disease or condition;    -   (l) An ocular disease or condition;    -   (m) A genital disease or condition, eg, a vaginal, labial,        penile or scrotal disease or condition;    -   (n) A sexually-transmissible disease or condition, eg,        gonorrhea, HIV infection, syphilis or Chlamydia infection;    -   (o) An ear disease or condition;    -   (p) A skin disease or condition;    -   (q) A heart disease or condition;    -   (r) A nasal disease or condition    -   (s) A haematological disease or condition, eg, anaemia, eg,        anaemia of chronic disease or cancer;    -   (t) A viral infection;    -   (u) A pathogenic bacterial infection;    -   (v) A cancer;    -   (w) An autoimmune disease or condition, eg, SLE;    -   (x) An inflammatory disease or condition, eg, rheumatoid        arthritis, psoriasis, eczema, asthma, ulcerative colitis,        colitis, Crohn's disease or IBD;    -   (y) Autism;    -   (z) ADHD;    -   (aa) Bipolar disorder;    -   (bb) ALS [Amyotrophic Lateral Sclerosis];    -   (cc) Osteoarthritis;    -   (dd) A congenital or development defect or condition;    -   (ee) Miscarriage;    -   (ff) A blood clotting condition;    -   (gg) Bronchitis;    -   (hh) Dry or wet AMD;    -   (ii) Neovascularisation (eg, of a tumour or in the eye);    -   (jj) Common cold;    -   (kk) Epilepsy;    -   (ll) Fibrosis, eg, liver or lung fibrosis;    -   (mm) A fungal disease or condition, eg, thrush;    -   (nn) A metabolic disease or condition, eg, obesity, anorexia,        diabetes, Type I or Type II diabetes.    -   (oo) Ulcer(s), eg, gastric ulceration or skin ulceration;    -   (pp) Dry skin;    -   (qq) Sjogren's syndrome;    -   (rr) Cytokine storm;    -   (ss) Deafness, hearing loss or impairment;    -   (tt) Slow or fast metabolism (ie, slower or faster than average        for the weight, sex and age of the subject);    -   (uu) Conception disorder, eg, infertility or low fertility;    -   (vv) Jaundice;    -   (ww) Skin rash;    -   (xx) Kawasaki Disease;    -   (yy) Lyme Disease;    -   (zz) An allergy, eg, a nut, grass, pollen, dust mite, cat or dog        fur or dander allergy;    -   (aaa) Malaria, typhoid fever, tuberculosis or cholera;    -   (bbb) Depression;    -   (ccc) Mental retardation;    -   (ddd) Microcephaly;    -   (eee) Malnutrition;    -   (fff) Conjunctivitis;    -   (ggg) Pneumonia;    -   (hhh) Pulmonary embolism;    -   (iii) Pulmonary hypertension;    -   (jjj) A bone disorder;    -   (kkk) Sepsis or septic shock;    -   (lll) Sinusitus;    -   (mmm) Stress (eg, occupational stress);    -   (nnn) Thalassaemia, anaemia, von Willebrand Disease, or        haemophilia;    -   (ooo) Shingles or cold sore;    -   (ppp) Menstruation;    -   (qqq) Low sperm count.        Neurodegenerative or CNS Diseases or Conditions for Treatment or        Prevention by the Method

In an example, the neurodegenerative or CNS disease or condition isselected from the group consisting of Alzheimer disease, geriopsychosis,Down syndrome, Parkinson's disease, Creutzfeldt-jakob disease, diabeticneuropathy, Parkinson syndrome, Huntington's disease, Machado-Josephdisease, amyotrophic lateral sclerosis, diabetic neuropathy, andCreutzfeldt Creutzfeldt-Jakob disease. For example, the disease isAlzheimer disease. For example, the disease is Parkinson syndrome.

In an example, wherein the method of the invention is practised on ahuman or animal subject for treating a CNS or neurodegenerative diseaseor condition, the method causes downregulation of Treg cells in thesubject, thereby promoting entry of systemic monocyte-derivedmacrophages and/or Treg cells across the choroid plexus into the brainof the subject, whereby the disease or condition (eg, Alzheimer'sdisease) is treated, prevented or progression thereof is reduced. In anembodiment the method causes an increase of IFN-gamma in the CNS system(eg, in the brain and/or CSF) of the subject. In an example, the methodrestores nerve fibre and/or reduces the progression of nerve fibredamage. In an example, the method restores nerve myelin and/or reducesthe progression of nerve myelin damage. In an example, the method of theinvention treats or prevents a disease or condition disclosed inWO2015136541 and/or the method can be used with any method disclosed inWO2015136541 (the disclosure of this document is incorporated byreference herein in its entirety, eg, for providing disclosure of suchmethods, diseases, conditions and potential therapeutic agents that canbe administered to the subject for effecting treatment and/or preventionof CNS and neurodegenerative diseases and conditions, eg, agents such asimmune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, anti-TIM3 orother antibodies disclosed therein).

Cancers for Treatment or Prevention by the Method

Cancers that may be treated include tumours that are not vascularized,or not substantially vascularized, as well as vascularized tumours. Thecancers may comprise non-solid tumours (such as haematological tumours,for example, leukaemias and lymphomas) or may comprise solid tumours.Types of cancers to be treated with the invention include, but are notlimited to, carcinoma, blastoma, and sarcoma, and certain leukaemia orlymphoid malignancies, benign and malignant tumours, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers andpaediatric tumours/cancers are also included.

Haematologic cancers are cancers of the blood or bone marrow. Examplesof haematological (or haematogenous) cancers include leukaemias,including acute leukaemias (such as acute lymphocytic leukaemia, acutemyelocytic leukaemia, acute myelogenous leukaemia and myeloblasts,promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronicleukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronicmyelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myeiodysplastic syndrome, hairy cell leukaemia andmyelodysplasia.

Solid tumours are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumours can be benign or malignant.Different types of solid tumours are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumours, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer,testicular tumour, seminoma, bladder carcinoma, melanoma, and CNStumours (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pineaioma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In an example, the cancer is a haematological cancer. In an example, thecancer is NSCLC. In an example, the cancer is renal cell carcinoma. Inan example, the cancer is urothelial carcinoma. In an example, thecancer is melanoma.

Autoimmune Diseases for Treatment or Prevention by the Method

-   -   Acute Disseminated Encephalomyelitis (ADEM)    -   Acute necrotizing hemorrhagic leukoencephalitis    -   Addison's disease    -   Agammaglobulinemia    -   Alopecia areata    -   Amyloidosis    -   Ankylosing spondylitis    -   Anti-GBM/Anti-TBM nephritis    -   Antiphospholipid syndrome (APS)    -   Autoimmune angioedema    -   Autoimmune aplastic anemia    -   Autoimmune dysautonomia    -   Autoimmune hepatitis    -   Autoimmune hyperlipidemia    -   Autoimmune immunodeficiency    -   Autoimmune inner ear disease (AIED)    -   Autoimmune myocarditis    -   Autoimmune oophoritis    -   Autoimmune pancreatitis    -   Autoimmune retinopathy    -   Autoimmune thrombocytopenic purpura (ATP)    -   Autoimmune thyroid disease    -   Autoimmune urticaria    -   Axonal & neuronal neuropathies    -   Balo disease    -   Behcet's disease    -   Bullous pemphigoid    -   Cardiomyopathy    -   Castleman disease    -   Celiac disease    -   Chagas disease    -   Chronic fatigue syndrome    -   Chronic inflammatory demyelinating polyneuropathy (CIDP)    -   Chronic recurrent multifocal ostomyelitis (CRMO)    -   Churg-Strauss syndrome    -   Cicatricial pemphigoid/benign mucosal pemphigoid    -   Crohn's disease    -   Cogans syndrome    -   Cold agglutinin disease    -   Congenital heart block    -   Coxsackie myocarditis    -   CREST disease    -   Essential mixed cryoglobulinemia    -   Demyelinating neuropathies    -   Dermatitis herpetiformis    -   Dermatomyositis    -   Devic's disease (neuromyelitis optica)    -   Discoid lupus    -   Dressler's syndrome    -   Endometriosis    -   Eosinophilic esophagitis    -   Eosinophilic fasciitis    -   Erythema nodosum    -   Experimental allergic encephalomyelitis    -   Evans syndrome    -   Fibromyalgia    -   Fibrosing alveolitis    -   Giant cell arteritis (temporal arteritis)    -   Giant cell myocarditis    -   Glomerulonephritis    -   Goodpasture's syndrome    -   Granulomatosis with Polyangiitis (GPA) (formerly called        Wegener's Granulomatosis)    -   Graves' disease    -   Guillain-Barre syndrome    -   Hashimoto's encephalitis    -   Hashimoto's thyroiditis    -   Hemolytic anemia    -   Henoch-Schonlein purpura    -   Herpes gestationis    -   Hypogammaglobulinemia    -   Idiopathic thrombocytopenic purpura (ITP)    -   IgA nephropathy    -   IgG4-related sclerosing disease    -   Immunoregulatory lipoproteins    -   Inclusion body myositis    -   Interstitial cystitis    -   Juvenile arthritis    -   Juvenile diabetes (Type 1 diabetes)    -   Juvenile myositis    -   Kawasaki syndrome    -   Lambert-Eaton syndrome    -   Leukocytoclastic vasculitis    -   Lichen planus    -   Lichen sclerosus    -   Ligneous conjunctivitis    -   Linear IgA disease (LAD)    -   Lupus (SLE)    -   Lyme disease, chronic    -   Meniere's disease    -   Microscopic polyangiitis    -   Mixed connective tissue disease (MCTD)    -   Mooren's ulcer    -   Mucha-Habermann disease    -   Multiple sclerosis    -   Myasthenia gravis    -   Myositis    -   Narcolepsy    -   Neuromyelitis optica (Devic's)    -   Neutropenia    -   Ocular cicatricial pemphigoid    -   Optic neuritis    -   Palindromic rheumatism    -   PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders        Associated with Streptococcus)    -   Paraneoplastic cerebellar degeneration    -   Paroxysmal nocturnal hemoglobinuria (PNH)    -   Parry Romberg syndrome    -   Parsonnage-Turner syndrome    -   Pars planitis (peripheral uveitis)    -   Pemphigus    -   Peripheral neuropathy    -   Perivenous encephalomyelitis    -   Pernicious anemia    -   POEMS syndrome    -   Polyarteritis nodosa    -   Type I, II, & III autoimmune polyglandular syndromes    -   Polymyalgia rheumatica    -   Polymyositis    -   Postmyocardial infarction syndrome    -   Postpericardiotomy syndrome    -   Progesterone dermatitis    -   Primary biliary cirrhosis    -   Primary sclerosing cholangitis    -   Psoriasis    -   Psoriatic arthritis    -   Idiopathic pulmonary fibrosis    -   Pyoderma gangrenosum    -   Pure red cell aplasia    -   Raynauds phenomenon    -   Reactive Arthritis    -   Reflex sympathetic dystrophy    -   Reiter's syndrome    -   Relapsing polychondritis    -   Restless legs syndrome    -   Retroperitoneal fibrosis    -   Rheumatic fever    -   Rheumatoid arthritis    -   Sarcoidosis    -   Schmidt syndrome    -   Scleritis    -   Scleroderma    -   Sjogren's syndrome    -   Sperm & testicular autoimmunity    -   Stiff person syndrome    -   Subacute bacterial endocarditis (SBE)    -   Susac's syndrome    -   Sympathetic ophthalmia    -   Takayasu's arteritis    -   Temporal arteritis/Giant cell arteritis    -   Thrombocytopenic purpura (TTP)    -   Tolosa-Hunt syndrome    -   Transverse myelitis    -   Type 1 diabetes    -   Ulcerative colitis    -   Undifferentiated connective tissue disease (UCTD)    -   Uveitis    -   Vasculitis    -   Vesiculobullous dermatosis    -   Vitiligo    -   Wegener's granulomatosis (now termed Granulomatosis with        Polyangiitis (GPA).        Inflammatory Diseases for Treatment or Prevention by the Method    -   Alzheimer's    -   ankylosing spondylitis    -   arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic        arthritis)    -   asthma    -   atherosclerosis    -   Crohn's disease    -   colitis    -   dermatitis    -   diverticulitis    -   fibromyalgia    -   hepatitis    -   irritable bowel syndrome (IBS)    -   systemic lupus erythematous (SLE)    -   nephritis    -   Parkinson's disease    -   ulcerative colitis.        Concepts:—

The invention provides the following Concepts.

-   -   1. A programmable nuclease for use in a method of treating an        acute microbial infection of a subject, wherein the microbial        infection is caused by microbes of a first species or strain and        the nuclease is programmable to cut a target site comprised by        the genomes of microbes that have infected the subject, whereby        microbes of the first species or strain are killed, or growth or        proliferation of the microbes is reduced, the treatment method        comprising exposing the subject to the nuclease wherein the        nuclease is programmed to cut the target site, whereby genomes        of the microbes comprised by the subject are cut and acute        microbial infection of the subject is treated.    -   2. A programmable nuclease for use in a method of durably        treating a microbial (eg, bacterial) infection of a subject,        wherein the microbial infection is caused by microbes of a first        species or strain and the nuclease is programmable to cut a        target site comprised by the genomes of microbes that have        infected the subject, whereby microbes of the first species or        strain are durably killed, or growth or proliferation of the        microbes is reduced, the treatment method comprising exposing        the subject to the nuclease wherein the nuclease is programmed        to cut the target site, whereby genomes of the microbes        comprised by the subject are cut and microbial infection of the        subject is durably treated.    -   3. The nuclease of Concept 2, wherein the nuclease (eg,        programmed nuclease) and/or a nucleic acid that programs the        nuclease to recognise and cut the target site is administered to        the subject at a first time (T1) and at a second time (T2)        wherein T2 is at least 1 hour after T1.    -   4. The nuclease of any preceding Concept, wherein the method        comprises reducing the infection at least 100-fold by the first        30 minutes (eg, by the first 15 minutes) of the treatment.    -   5. The nuclease of any preceding Concept, wherein the method        comprises maintaining reduction of the infection by at least        100-fold for at least 60 minutes (eg, at least 120 minutes)        after exposing the subject to the programmed nuclease.    -   6. The nuclease of any preceding Concept, wherein the method        comprises reducing the infection such that the reduction in        infection persists for 30 minutes immediately after the first 30        minutes of the treatment.    -   7. The nuclease of any preceding Concept, wherein the method        comprises administering to the subject a RNA or a nucleic acid        that encodes an RNA for expression of the RNA in the subject,        wherein the RNA complexes with the nuclease to program the        nuclease to cut the target site in microbes comprised by the        subject.    -   8. The nuclease of Concept 7, wherein the nuclease is        administered simultaneously or sequentially with the RNA or        nucleic acid to the subject.    -   9. The nuclease of Concept 7, wherein the subject comprises the        nuclease prior to administration of the RNA or nucleic acid to        the subject.    -   10. The nuclease of any one of Concepts 7 to 9, wherein a        plurality of viruses (eg, phage) are administered to the        subject, wherein each virus comprises a copy of the nucleic        acid, wherein the viruses infect the microbes comprised by the        subject to deliver thereto the nucleic acid.    -   11. The nuclease of Concept 10, wherein the ratio of        administered viruses:microbes comprised by the subject is from        10 to 150.    -   12. The nuclease according to any preceding Concept, wherein the        subject is a human or animal, optionally wherein the subject is        a human over 65 years of age or is a paediatric patient.    -   13. The nuclease according to Concept 12, wherein the infection        is an infection of the lungs, abdomen or urinary tract; or        wherein the subject has undergone surgery, is on an        immunosuppressant medication and/or is suffering from a chronic        disease.    -   14. The nuclease according to any preceding Concept, wherein the        infection is reduced by at least 90% for 1 hour or more,        optionally by the first 30 minutes (eg, by the first 15 minutes)        of the treatment.    -   15. The nuclease according to any preceding Concept, wherein the        method comprises reducing the infection at least 100-fold by the        first 30 minutes (eg, by the first 15 minutes) of the treatment;        and wherein reduction of the infection by at least 100-fold is        maintained for at least 60 minutes (eg, at least 120, 145 or 180        minutes) after exposing the subject to the programmed nuclease.    -   16. The nuclease according to any one of Concepts 12 to 15,        wherein the method treats or prevents septicaemia and/or sepsis        (eg, septic shock) in the subject.    -   17. The nuclease of Concept 16, wherein at the start of the        treatment, the subject (eg, a human) has a temperature of        <36° C. or >38° C.; a heart rate of >90/min, a respiratory rate        of breaths/min or PaCO₂<43 kPa; and white blood cell count of        <4000/mm³ or >12,000/mm³.    -   18. The nuclease of Concept 16 or 17, wherein at the start of        the treatment, the subject (eg, a human) has presence of two or        more of the following: abnormal body temperature, abnormal heart        rate, abnormal respiratory rate, abnormal blood gas and abnormal        white blood cell count.    -   19. The nuclease of any preceding Concept, wherein the subject        is a human or animal and the microbes are bacteria (eg, E. coli        or C. dificile), wherein blood infection of the subject by the        bacteria is reduced at least 100- or 1000-fold by the first 30        minutes (eg, by the first 15 minutes) of the treatment.    -   20. The nuclease of any one of Concepts 12 to 19, wherein the        blood of the subject is infected with from 10⁷ to 10¹² CFU/ml of        the bacteria immediately before the treatment.    -   21. The nuclease according to any one of Concepts 1 to 11,        wherein the subject is a plant.    -   22. The nuclease according to any preceding Concept, wherein the        microbes are bacteria.    -   23. The nuclease according to Concept 22, wherein the bacteria        are gram positive bacteria.    -   24. The nuclease according to Concept 22 or 23, wherein the        bacteria are Staphylococcus, Streptococcus, Enterococcus,        Legionella, Heamophilus, Ghonnorhea, Acinetobacter, Escherichia,        Klebsiella, Pseudomonas or Stenotrophomonas bacteria (eg, E.        coli (eg, EHEC E. coli), C. dificile, V. cholera, Staphylococcus        (eg, S. aureus or MRSA), Streptococcus pyogenes, Acinetobacter        baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella        pneumoniae bacteria).    -   25. The nuclease according to any preceding Concept, wherein the        nuclease is a Cas nuclease (eg, a Cas 3 or 9), a meganuclease, a        TALEN (Transcription activator-like effector nuclease) or zinc        finger nuclease.    -   26. A plurality of viruses (eg, phage or phagemids for producing        phage) for use with the nuclease of any preceding Concept in the        method of treatment, wherein each virus comprises a copy of a        nucleic acid as defined in any one of Concepts 7 to 9, wherein        the viruses are capable of infecting microbes comprised by the        subject to deliver thereto the nucleic acid.    -   27. A composition comprising a plurality of nucleic acids for        programming the nuclease of any one of Concepts 1 to 25 in the        method of treatment, wherein each nucleic acid is a nucleic acid        as defined in any one of Concepts 7 to 9.    -   28. A CRISPR/Cas system comprising a nuclease according to any        preceding Concept for use in the method of treatment, wherein        the nuclease is a Cas nuclease (eg, a Cas 3 or 9) and the system        comprises one or more guide RNAs or DNA encoding one or more        guide RNAs, wherein each guide RNA is capable of programming the        Cas nuclease to cut a target site comprised by the genomes of        the microbes.    -   29. A guide RNA or a DNA encoding a guide RNA for use in the        system of Concept 28 for use in the method of treating an acute        microbial infection in the subject, eg, septicaemia or sepsis.    -   30. A nucleic acid vector comprising the guide RNA or DNA        recited in Concept 27 or 29.    -   31. The vector of Concept 30 wherein the vector is a phage,        phagemid, viriophage, virus, plasmid (eg, conjugative plasmid)        or transposon.    -   32. An anti-sepsis or anti-septicaemia composition for        administration to a human or animal for treating sepsis or        septicaemia, the composition comprising a plurality of vectors,        wherein each vector is according to Concept 30 or 31.    -   33. A method of treating an acute microbial infection of a        subject, wherein the method is as defined by any preceding        Concept.    -   34. Use of a nuclease, plurality of viruses, system, guide RNA,        DNA or vector of any one of Concepts 1 to 26 and 28 to 30, in        the manufacture of a composition for carrying out a method of        treatment as defined by any preceding Concept, wherein the        subject is an organism other than a human or animal.    -   35. Use of a nuclease, plurality of viruses, system, guide RNA,        DNA or vector of any one of Concepts 1 to 26 and 28 to 30, in        the manufacture of a composition for carrying out an ex vivo        method of treatment of a microbial infection of a substrate,        wherein the microbial infection is caused by microbes of a first        species or strain and the nuclease is programmable to cut a        target site comprised by the genomes of microbes that have        infected the substrate, whereby microbes of the first species or        strain are killed, or growth or proliferation of the microbes is        reduced, the treatment method comprising exposing the subject to        the nuclease wherein the nuclease is programmed to cut the        target site, whereby genomes of the microbes comprised by the        subject are cut and acute microbial infection of the substrate        is treated.    -   36. Use of a programmable nuclease in the manufacture of a        composition for carrying out an ex vivo method of treatment of a        microbial infection of a substrate, wherein the microbial        infection is caused by microbes of a first species or strain and        the nuclease is programmable to cut a target site comprised by        the genomes of microbes that have infected the substrate,        whereby microbes of the first species or strain are killed, or        growth or proliferation of the microbes is reduced, the        treatment method comprising exposing the subject to the nuclease        wherein the nuclease is programmed to cut the target site,        whereby genomes of the microbes comprised by the subject are cut        and acute microbial infection of the substrate is treated.    -   37. The use of Concept 34, 35 or 36, wherein the nuclease (eg,        programmed nuclease) and/or a nucleic acid that programs the        nuclease to recognise and cut the target site is administered to        the subject or substrate at a first time (T1) and at a second        time (T2) wherein T2 is at least 1 hour after T1.    -   38. The use of any one of Concepts 34 to 37, wherein the        infection is reduced at least 100-fold by the first 30 minutes        (eg, by the first 15 minutes) of the treatment.    -   39. The use of any one of Concepts 34 to 38, wherein the        reduction of the infection is maintained by at least 100-fold        for at least 60 minutes (eg, at least 120 minutes) after        exposing the subject to the programmed nuclease.    -   40. The use of any one of Concepts 34 to 39, wherein the        reduction in infection persists for 30 minutes immediately after        the first 30 minutes of the treatment.    -   41. The use of any one of Concepts 34 to 40, wherein the method        comprises administering to the subject or substrate a RNA or a        nucleic acid that encodes an RNA for expression of the RNA in or        on the subject or substrate, wherein the RNA complexes with the        nuclease to program the nuclease to cut the target site in        microbes comprised by the subject or substrate.    -   42. The use of Concept 41, wherein the nuclease is administered        simultaneously or sequentially with the RNA or nucleic acid to        the subject or substrate.    -   43. The use of Concept 41, wherein the subject or substrate        comprises the nuclease prior to administration of the RNA or        nucleic acid.    -   44. The use of any one of Concepts 41 to 43, wherein a plurality        of viruses (eg, phage) are administered to the subject or        substrate, wherein each virus comprises a copy of the nucleic        acid, wherein the viruses infect the microbes comprised by the        subject or substrate to deliver thereto the nucleic acid.    -   45. The use of Concept 44, wherein the ratio of administered        viruses:microbes is from 10 to 150.    -   46. The use of any one of Concepts 34 to 45, wherein the        infection is reduced by at least 90% for 1 hour or more,        optionally by the first 30 minutes (eg, by the first 15 minutes)        of the treatment.    -   47. The use of any one of Concepts 34 to 46, wherein the        infection is reduced at least 100-fold by the first 30 minutes        (eg, by the first 15 minutes) of the treatment; and wherein        reduction of the infection by at least 100-fold is maintained        for at least 60 minutes (eg, at least 120, 145 or 180 minutes)        after exposing the subject or substrate to the programmed        nuclease.    -   48. The use of any one of Concepts 34 to 47, wherein the subject        is a plant; or wherein the substrate is a metallic, plastic,        concrete, stone, wood, glass or ceramic substrate.    -   49. The use of any one of Concepts 34 to 48, wherein the        microbes are bacteria.    -   50. The use according to Concept 49, wherein the bacteria are        gram positive bacteria.    -   51. The use according to Concept 49 or 50, wherein the bacteria        are Staphylococcus, Streptococcus, Enterococcus, Legionella,        Heamophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella,        Pseudomonas or Stenotrophomonas bacteria (eg, E. coli (eg,        EHEC E. coli), C. dificile, V. cholera, Staphylococcus (eg, S.        aureus or MRSA), Streptococcus pyogenes, Acinetobacter        baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella        pneumoniae bacteria).    -   52. The use of any one of Concepts 34 to 51, wherein the        nuclease is a Cas nuclease (eg, a Cas 3 or 9), a meganuclease, a        TALEN (Transcription activator-like effector nuclease) or zinc        finger nuclease.

EMBODIMENTS

-   -   1. A method for treating a pathogenic bacterial infection in a        human or animal subject caused by bacteria (first bacteria) of a        first species or strain, the method comprising selectively        killing first bacteria comprised by the subject by cutting a        target site comprised by the genomes of the first bacteria,        wherein the cutting is carried out using a programmable nuclease        that is programmed to cut the target site, wherein the subject        is suffering from a further disease or condition other than the        pathogenic bacterial infection and the method comprises        administering a therapy to the subject for treating or        preventing the further disease or condition, wherein the        nuclease treats the infection and the therapy is efficacious in        the presence of the programmed nuclease to treat or prevent the        disease or condition.    -   2. The method of Embodiment 1, wherein the subject is a cancer        patient and the therapy comprises administration of a        haematopoietic stem cell transplant, chemotherapeutic agent,        immune checkpoint inhibitor, immune checkpoint agonist or an        immune cell enhancer; adoptive cell therapy; radiation or        surgery.    -   3. The method of Embodiment 2, wherein the therapy is an immune        checkpoint inhibitor antibody, or an antibody selected from        ipilimumab (or YERVOY®), tremelimumab, nivolumab (or OPDIVO®),        pembrolizumab (or KEYTRUDA®), pidilizumab, BMS-936559,        durvalumab (or IMFINZI®) and atezolizumab (or TECENTRIQ®).    -   4. The method of Embodiment 1, wherein the therapy is a tissue,        organ or cell transplant.    -   5. The method of Embodiment 1, wherein the treatment of the        bacterial infection is carried out simultaneously with the        administration of the therapy to the subject.    -   6. The method of Embodiment 1, wherein the treatment of the        bacterial infection is carried out immediately before or after        administering the therapy to the subject.    -   7. The method of Embodiment 1, wherein the method comprises        administering to the subject a or a nucleic acid that encodes an        RNA for expression of the RNA in the subject, wherein the RNA        complexes with the nuclease to program the nuclease to cut the        target site in first bacteria comprised by the subject, thereby        killing the first bacteria.    -   8. The method of Embodiment 1, comprising administering a        nucleic acid vector to the subject, wherein the vector encodes        the programmable nuclease.    -   9. The method of Embodiment 1, wherein the programmable nuclease        is an endogenous nuclease of the first cells.    -   10. The method of Embodiment 1, wherein the efficacy of the        therapy in the presence of the programmed nuclease is greater        than the efficacy of the therapy in the presence of a        broad-spectrum antibiotic.    -   11. The method of Embodiment 1, wherein the efficacy of the        therapy in the presence of the programmed nuclease is greater        than the efficacy of the therapy in the presence of an        antibiotic selected from methicillin, vancomycin, linezolid,        daptomycin, quinupristin, dalfopristin; teicoplanin;        cephalosporin; carbapenem; fluoroquinolone; aminoglycoside;        colistin; erythromycin; clindamycin; beta-lactam; macrolide;        amoxicillin; azithromycin; penicillin; ceftriaxone;        azithromycin; ciprofloxacin; isoniazid (INH); rifampicin (RMP);        amikacin; kanamycin; capreomycin; trimethoprim; itrofurantoin;        cefalexin; amoxicillin; metronidazole (MTZ); cefixime;        tetracycline; and meropenem.    -   12. The method of Embodiment 1, wherein the first bacteria is        selected from (i) Staphylococcus aureus that is resistant to an        antibiotic selected from methicillin, vancomycin, linezolid,        daptomycin, quinupristin, dalfopristin and teicoplanin; (ii)        Pseudomonas aeuroginosa that is resistant to an antibiotic        selected from cephalosporins, carbapenems, fluoroquinolones,        aminoglycosides and colistin; (iii) Klebsiella species that is        resistant to carbapenem; (iv) Streptoccocus species that is        resistant to an antibiotic selected from erythromycin,        clindamycin, beta-lactam, macrolide, amoxicillin, azithromycin        and penicillin; (v) Salmonella species that is resistant to an        antibiotic selected from ceftriaxone, azithromycin and        ciprofloxacin; (vi) Shigella species that is resistant to        ciprofloxacin or azithromycin; (vii) Mycobacterium tuberculosis        that is resistant to an antibiotic selected from Resistance to        isoniazid (INH), rifampicin (RMP), fluoroquinolone, amikacin,        kanamycin, capreomycin and azithromycin; (viii) Enterococcus        species that is resistant to vancomycin; (ix) Enterobacteriaceae        species that is resistant to an antibiotic selected from        cephalosporin and carbapenem; (x) E. coli that is resistant to        an antibiotic selected from trimethoprim, itrofurantoin,        cefalexin and amoxicillin; (xi) Clostridium species that is        resistant to metronidazole (MTZ), fluoroquinolone or        carbapenem; (xii) Neisseria gonnorrhoea that is resistant to an        antibiotic selected from cefixime, ceftriaxone, azithromycin and        tetracycline; (xiii) Acinetoebacter baumannii that is resistant        to an antibiotic selected from beta-lactam, meropenem and        carbapenem; and (xiv) Campylobacter species that is resistant to        ciprofloxacin or azithromycin.    -   13. The method of Embodiment 1, wherein the treatment of the        infection treats or prevents in the subject a condition selected        from vaginosis, meningitis, pneumonia, urinary tract infection,        cystitis, nephritis, gastroenteritis, a skin infection,        impetigo, erysipelas, cellulitis, septicaemia or sepsis in the        subject.    -   14. The method of Embodiment 1, wherein the further disease or        condition is a cancer; autoimmune disease or condition; or GI        tract disease or condition.    -   15. The method of Embodiment 1, wherein the subject comprises        bacteria (second bacteria) of one or more strains or species        that are different to the first strain or species, wherein the        genomes of the second bacteria do not comprise the target site,        wherein the genomes of the second bacteria are not cut by the        programmed nuclease in the subject, whereby second bacteria        survive in the presence of the programmed nuclease in the        patient; and wherein the therapy is efficacious in the presence        of the second bacteria.    -   16. The method of Embodiment 15, wherein reduction in the second        bacteria in patients is associated with reduced efficacy of the        therapy.    -   17. The method of Embodiment 15, wherein the second bacteria are        selected from the group consisting of Akkermansia, Alistipes,        Bacteroides, Barnesiella, Bifidobacterium, Clostridium,        Collinsella, Enterococcus, Fusobacterium, Lactobacillus,        Propionibacterium, Ruminococcus, Segmented filamentous bacteria        (SFB); Veillonella, Prevotella, Escherichia and Streptococcus        bacteria.    -   18. The method of Embodiment 1, wherein the first bacteria are        selected from the group consisting of E. coli, C. dificile, V.        cholera, Staphylococcus, Streptococcus pyogenes, Acinetobacter        baumannii, Legionella, Pseudomonas aeruginosa and Klebsiella        pneumoniae bacteria.    -   19. The method of Embodiment 1, wherein the nuclease is a Cas        nuclease, a meganuclease, a Transcription activator-like        effector nuclease (TALEN) or zinc finger nuclease.    -   20. A method for treating a pathogenic bacterial infection in a        cancer patient caused by bacteria (first bacteria) of a first        species or strain, the method comprising selectively killing        first bacteria comprised by the subject by cutting a target site        comprised by the genomes of the first bacteria, wherein the        cutting is carried out using a Cas nuclease that is programmed        by guide RNA to cut the target site, wherein the method        comprises administering an immunotherapy to the subject for        treating cancer in the patient, wherein the nuclease treats the        infection and the immunotherapy is efficacious in the presence        of the programmed nuclease to treat the cancer.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications and all US equivalentpatent applications and patents are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference. The use of the word “a” or “an” when used in conjunction withthe term “comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of “one or more,” “atleast one,” and “one or more than one.” The use of the term “or” in theclaims is used to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” or similar as used herein refers toall permutations and combinations of the listed items preceding theterm. For example, “A, B, C, or combinations thereof is intended toinclude at least one of: A, B, C, AB, AC, BC, or ABC, and if order isimportant in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC,or CAB. Continuing with this example, expressly included arecombinations that contain repeats of one or more item or term, such asBB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilledartisan will understand that typically there is no limit on the numberof items or terms in any combination, unless otherwise apparent from thecontext.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting Examples.

EXAMPLES

Precision Fast Bacteria Killing with Programmable Nucleases

The examples provide a method for fast and precision killing ofEscherichia coli and Clostridium dificile strains. As a modelprogrammable nuclease system, we used a CRISPR guided vector (CGV™)system to specifically target enterohemorrhagic E. coli (EHEC) andprobiotic coli Nissle.

Example 1. Precision Killing of Target Strain Enterohemorrhagic E. coli(EHEC)

1.1. Design, Construction and Delivery of CRISPR Guided Vector (CGV)System Targeting E. coli (EHEC) ATCC43888.

The invention provides a CGV system to specifically targetenterohemorrhagic E. coli (EHEC) ATCC43888 (a human fecal isolateobtained from the American Type Culture Collection). The CGV systemcomprises two vectors: (a) a vector containing a tracrRNA and the Cas9protein from Streptococcus pyogenes (SpCas); (b) a vector containing aguide RNA (gRNA) that comprises a nucleotide sequence capable ofhybridizing to a target sequence in the host cells to guide SpCas9 tothe target sequence. To enable specific killing of E. coli (EHEC)ATCC43888, a particular sequence from the genome of this strain waschosen to target. Specifically, the sequence contains 20 nucleotidesfrom the 23S ribosomal RNA gene from E. coli (EHEC) ATCC43888.Additionally, the 5′-NGG protospacer adjacent motif (PAM) was locatedadjacent to the selected target sequence. The selected target sequencein the 23S rRNA gene can be found in Table 3.

1.2 Characterization of the CGV System Targeting E. coli (EHEC)ATCC43888.

To establish the CGV system functionality in mediating sequence-specifickilling in E. coli (EHEC) ATCC43888, the system was transformed into E.coli (EHEC) ATCC43888 cells. Overnight cultures were diluted 1:100 infresh lysogeny broth (LB) and grown to mid-exponential phase OD600 ˜0.6.The CRISPR system was induced by adding theophylline and arabinose (2 mMtheophylline and 1% arabinose), and survival of the strain was followedover time by plating the cultures in serial dilutions every 15 minutes,for 1 h (FIG. 1B). CRISPR induction in E. coli (EHEC) surprisinglytriggered a rapid killing of the cells, achieving 99.98% killing within30 minutes of induction (FIG. 1A).

Example 2. In Vivo CRISPR Killing of Target Strain Enterohemorrhagic E.coli (EHEC) in Galleria mellonella Larvae In Vivo Infection Model

2.1. CRISPR Efficacy Against E. coli (EHEC) ATCC43888 Infections inGalleria mellonella

CRISPR killing of target strain E. coli (EHEC) ATCC43888 was tested inG. mellonella in vivo infection model. To this aim, G. mellonella larvaewere delivered injections of bacteria 10⁸ CFU E. coli (EHEC) ATCC43888)behind the final left proleg. Approximately 1 h after the injection,CRISPR inducers (2 mM theophylline and 1% arabinose) were administeredbehind the final right proleg. Larvae were incubated at 37° C. and theywere sacrificed after 1 and 2 h after induction. As shown in FIGS. 2 and3 , CRISPR induction killed 99% of the population after 2 h, as comparedto the off-target control.

2.2. Survival Curves of G. mellonella Larvae Infected withEnterohemorrhagic E. coli (EHEC).

G. mellonella larvae were delivered injections of bacteria (8×10⁴ CFU E.coli ATCC43888) behind the final left proleg. Approximately 1 h afterthe injection, CRISPR inducers (2 mM theophylline and 1% arabinose) wereadministered behind the final right proleg. Larvae were incubated at 37°C. and survival was monitored for 115 h, with death indicated by lack ofmovement and unresponsiveness to touch. CRISPR killing of target strainE. coli (EHEC) ATCC43888 in G. mellonella larvae significantly improvedsurvival of the larvae compared to the off-target control (FIG. 4 )(log-rank test, P<0.03).

Example 3. Precision Killing of Target Strain Probiotic E. coli Nissle1917

3.1. Design, Construction and Delivery of CRISPR Guided Vector (CGV)System Targeting E. coli Nissle 1917.

The invention provides a CGV system to specifically target E. coliNissle 1917. The CGV system comprises two vectors: (a) a vectorcontaining a tracrRNA and the Cas9 from Streptococcus pyogenes (SpCas);(b) a vector containing a guide RNA (gRNA) that comprises a nucleotidesequence capable of hybridizing to a target sequence in the host cellsto guide SpCas9 to the target sequence. To enable specific killing of E.coli Nissle 1917, a specific sequence from the genome of this strain waschosen to target. Specifically, the sequence contains 20 nucleotidesfrom the pks gene from E. coli Nissle 1917. Additionally, the 5′-NGGprotospacer adjacent motif (PAM) was located adjacent to the selectedtarget sequence. The selected target sequence in the pks gene can befound in Table 3.

Furthermore, a different genome target was selected to specifically killE. coli Nissle 1917. The sequence contains 20 nucleotides from the yapHgene. Additionally, the 5′-NGG protospacer adjacent motif (PAM) waslocated adjacent to the selected target sequence. The selected targetsequence in yapH gene can be found in Table 3.

3.2. Construction and Delivery of CRISPR Guided Vectors (CGV) TargetingE. coli Nissle 1917

To establish CGVs functionality in mediating sequence-specific killingin E. coli Nissle 1917, the CGV system was transformed into E. coliNissle 1917 cells. Overnight cultures were diluted 1:100 in freshlysogeny broth (LB) and grown to mid-exponential phase OD600 ˜0.6. TheCRISPR system was induced by adding theophylline and arabinose (2 mMtheophylline and 1% arabinose), and survival of the strain was followedover time by plating the cultures in serial dilutions every 15 minutes,for 3 h (FIGS. 5B and 6B). FIGS. 5B and 6A show CRISPR killing assay inE. coli Nissle 1917, targeting pks gene and yapH gene, respectively. Inboth cases, CRISPR induction triggers a rapid killing of E. coli Nissle1917 cells, achieving 99.98% killing within only 15 minutes ofinduction.

Example 4. In Vitro CRISPR Killing of Clostridium difficile byConjugative Plasmid Vectors and Cas3

This experiment involves the precision killing of Clostridium difficileusing a gRNA-encoding CRISPR array that is delivered from a probioticcarrier bacterial species by conjugative plasmids as vectors (which wecall CRISPR guided vectors (CGV™)). A carrier bacterium (E. coli donorstrain containing the CRISPR guided vector (CGV™)) was mated withClostridium difficile which was killed upon delivery of the CGV™containing the designed array. This CGV™ harnessed the endogenous Cas3machinery of Clostridium difficile 630Δerm. A 100% killing ofClostridium difficile cells was achieved.

Introduction

Clostridium difficile (C. difficile) is a spore-forming humanopportunistic pathogen that can asymptomatically colonize the intestineof healthy individuals. The two main risk factors for contracting C.difficile-associated diseases, such as nosocomial diarrhea, are age andantibiotic treatment and can have fatal consequences. C. difficile630Δerm, the subject of our study, is a well-characterized strain and itis widely used for the generation of mutant specimens.

Study Objectives

Objective 1: Delivery of CGVs by Conjugation.

A CRISPR guided vector (CGV) containing an array to specifically targetand kill C. difficile was designed and assembled. The same CGV lackingthe array was assembled to use as a control for conjugation efficiency.Both CGVs were transformed into the carrier strain Escherichia coliCA434, which was used as a donor strain to conjugate the plasmid intoour strain of interest C. difficile 630Δerm.

Objective 2: Harnessing Clostridium difficile Endogenous Cas3 Machinery.

Upon transcription of the delivered CRISPR array in the recipient targetstrain C. difficile, the endogenous Cas3 was guided to cut its own DNA;leading to bacterial death.

Objective 3: Eradication of Clostridium difficile 630Δerm.

Achievement of efficient killing of transconjugant C. difficile cellsusing designed CGVs.

Materials and Methods

Bacterial Strains and Growth Conditions

E. coli strain CA434 was acquired from Chain Biotech. It was cultured onnutrient-rich media (2×YT) and grown overnight at 37° C. and 250 rpm.Medium was supplemented with 12.5 μg/mL of thiamphenicol when requiredto maintain the CGVs.

Clostridium difficile 630Δerm was grown on BHI agar supplemented with 5g/L of yeast extract, 0.03% L-cystein, 250 ug/ml D-cycloserine and 8ug/ml of cefoxitin (BHIS+CC). C. difficile was grown overnight in a Coyvinyl anaerobic cabinet in an atmosphere of 92% N₂, 6% CO₂ and 2% H2 at37° C. The mating of the donor CA434 and C. difficile was grown on plainBHI agar to allow for growth of the donor strain. Thiamphenicol wasadded to BHIS+CC plates to a final concentration of 12.5 μg/mL forselection of transconjugants after mating. All plates were dried for 1.5hours and transferred, along with the broth version of this medium, tothe anaerobic chamber at least 3 hours before use.

CGV Transfer Procedures

Carrier cells of E. coli CA434 were obtained by electroporation ofeither of our CGVs (control vector pMTL84151-FJ797649 and CRISPR vectorpMTL84151-cdCRISPR1). In order to do that, overnight cultures of E. coliCA434 were diluted 1:100 in fresh 2×YT medium without selection andgrown to OD600 ˜0.5. Then, they were made electrocompetent by standardprocedures (Sharan et al., 2009). Electrocompetent cells weretransformed with either plasmid pMTL84151-FJ797649 orpMTL84151-cdCRISPR1 and recovered in 2×YT for 1 h at 37° C. with shaking(250 rpm). Finally, they were plated on LB agar supplemented with 12.5μg/mL thiamphenicol for selection of transformants. Transformants weregrown in liquid 2×YT supplemented with 12.5 μg/mL thiamphenicol at 37°C. and 250 rpm for mating with C. difficile. 1 ml of donor cells wascentrifuged at 4000×g for 2 minutes, supernatant removed and carefullywashed with 400 μl of PBS. After a second centrifugation cycle thepellet was transferred to the anaerobic chamber for mating with C.difficile in BHI non-selective plates. C. difficile was prepared formating following a modified protocol (Des Purdy et al., 2002). C.difficile 630Δerm was incubated overnight in selective BHIS+CC plates,from which, a scrape was inoculated overnight in 1 ml of non-selectiveBHI and incubated over night for mating. 200 μl of that culture was usedto resuspend the pelleted donor cells and mixed culture was plated in 20μl spots on top of non-selective BHI plates. The mating was incubated24h to allow for conjugation. After incubation, the whole plate wasthoroughly scraped with a sterile inoculation loop, resuspended in BHIand serial dilutions were plated on BHI+CC plates to prevent growth ofdonor E. coli and on BHI+CC supplemented with thiamphenicol foradditional selection of transconjugants. Single colonies were countedafter 48 hours.

Results

Replicates of BHI+CC+Thiamphenicol plates, selecting for C. difficiletransconjugants carrying the control CGV, showed a consistent number ofcolonies resulting in about ˜600-750 CFUs per mating experiment. For themating of C. difficile with E. coli CA434 carrying the CGV with theCRISPR array the plates were empty, no colonies were observed. Thistranslates into 100% killing of transconjugant C. difficile 630Δermcells receiving the CRISPR array (see FIG. 7 : Killing of transconjugantC. difficile 630Δerm).

DISCUSSION AND CONCLUSIONS

The results of this experiment show that we could successfully conjugateCGVs containing the desired CRISPR arrays into C. difficile 630Δerm froman E. coli carrier bacterium. We could also successfully harness C.difficile endogenous Cas3 machinery for very efficient CRISPR killing.

REFERENCES

-   Purdy D, O'Keeffe T A, Elmore M, Herbert M, McLeod A, Bokori-Brown    M, Ostrowski A, Minton N P. (2002) Conjugative transfer of    clostridial shuttle vectors from Escherichia coli to Clostridium    difficile through circumvention of the restriction barrier. Molec.    Microbiology 46(2), 439-452-   Sharan, S. K., Thomason, L. C., Kuznetsov, S. G., and    Court, D. L. (2009) Recombineering: a homologous recombination-based    method of genetic engineering. Nat. Protoc. 4, 206-223

TABLE 1 Example Bacteria Optionally, the bacteria are selected from thisTable. Abiotrophia Acidocella Actinomyces Alkalilimnicola AquaspirillumAbiotrophia Acidocella Actinomyces bovis Alkalilimnicola Aquaspirillumdefectiva aminolytica Actinomyces ehrlichii polymorphum AcaricomesAcidocella facilis denticolens Alkaliphilus Aquaspirillum AcaricomesAcidomonas Actinomyces Alkaliphilus putridiconchylium phytoseiuliAcidomonas europaeus oremlandii Aquaspirillum Acetitomaculum methanolicaActinomyces Alkaliphilus serpens Acetitomaculum Acidothermus georgiaetransvaalensis Aquimarina ruminis Acidothermus ActinomycesAllochromatium Aquimarina Acetivibrio cellulolyticus gerencseriaeAllochromatium latercula Acetivibrio Acidovorax Actinomyces vinosumArcanobacterium cellulolyticus Acidovorax hordeovulneris AlloiococcusArcanobacterium Acetivibrio anthurii Actinomyces Alloiococcus otitishaemolyticum ethanolgignens Acidovorax caeni howellii AllokutzneriaArcanobacterium Acetivibrio Acidovorax Actinomyces Allokutzneria albatapyogenes multivorans cattleyae hyovaginalis AltererythrobacterArchangium Acetoanaerobium Acidovorax citrulli ActinomycesAltererythrobacter Archangium Acetoanaerobium Acidovorax israeliiishigakiensis gephyra noterae defluvii Actinomyces Altermonas ArcobacterAcetobacter Acidovorax johnsonii Altermonas Arcobacter butzleriAcetobacter aceti delafieldii Actinomyces haloplanktis ArcobacterAcetobacter Acidovorax facilis meyeri Altermonas cryaerophiluscerevisiae Acidovorax Actinomyces macleodii Arcobacter Acetobacterkonjaci naeslundii Alysiella halophilus cibinongensis AcidovoraxActinomyces neuii Alysiella crassa Arcobacter Acetobacter temperansActinomyces Alysiella filiformis nitrofigilis estunensis Acidovoraxodontolyticus Aminobacter Arcobacter Acetobacter valerianellaeActinomyces oris Aminobacter skirrowii fabarum Acinetobacter Actinomycesaganoensis Arhodomonas Acetobacter Acinetobacter radingae AminobacterArhodomonas ghanensis baumannii Actinomyces aminovorans aquaeoleiAcetobacter Acinetobacter slackii Aminobacter Arsenophonus indonesiensisbaylyi Actinomyces niigataensis Arsenophonus Acetobacter Acinetobacterturicensis Aminobacterium nasoniae lovaniensis bouvetii ActinomycesAminobacterium Arthrobacter Acetobacter Acinetobacter viscosus mobileArthrobacter agilis malorum calcoaceticus Actinoplanes AminomonasArthrobacter albus Acetobacter Acinetobacter Actinoplanes AminomonasArthrobacter nitrogenifigens gerneri auranticolor paucivorans aurescensAcetobacter oeni Acinetobacter Actinoplanes Ammoniphilus ArthrobacterAcetobacter haemolyticus brasiliensis Ammoniphilus chlorophenolicusorientalis Acinetobacter Actinoplanes oxalaticus ArthrobacterAcetobacter johnsonii consettensis Ammoniphilus citreus orleanensisAcinetobacter junii Actinoplanes oxalivorans Arthrobacter AcetobacterAcinetobacter deccanensis Amphibacillus crystallopoietes pasteurianuslwoffi Actinoplanes Amphibacillus Arthrobacter Acetobacter Acinetobacterderwentensis xylanus cumminsii pornorurn parvus Actinoplanes AmphriteaArthrobacter Acetobacter Acinetobacter digitatis Amphritea balenaeglobiformis senegalensis radioresistens Actinoplanes Amphritea japonicaArthrobacter Acetobacter Acinetobacter durhamensis Amycolatopsishistidinolovorans xylinus schindleri Actinoplanes Amycolatopsis albaArthrobacter ilicis Acetobacterium Acinetobacter soli ferrugineusAmycolatopsis Arthrobacter luteus Acetobacterium AcinetobacterActinoplanes albidoflavus Arthrobacter bakii tandoii globisporusAmycolatopsis methylotrophus Acetobacterium Acinetobacter Actinoplanesazurea Arthrobacter carbinolicum tjernbergiae humidus Amycolatopsismysorens Acetobacterium Acinetobacter Actinoplanes coloradensisArthrobacter dehalogenans towneri italicus Amycolatopsis nicotianaeAcetobacterium Acinetobacter Actinoplanes lurida Arthrobacter fimetariumursingii liguriensis Amycolatopsis nicotinovorans AcetobacteriumAcinetobacter Actinoplanes mediterranei Arthrobacter malicum venetianuslobatus Amycolatopsis oxydans Acetobacterium Acrocarpospora Actinoplanesrifamycinica Arthrobacter paludosum Acrocarpospora missouriensisAmycolatopsis pascens Acetobacterium corrugata Actinoplanes rubidaArthrobacter tundrae Acrocarpospora palleronii Amycolatopsisphenanthrenivorans Acetobacterium macrocephala Actinoplanes sulphureaArthrobacter wieringae Acrocarpospora philippinensis Amycolatopsispolychromogenes Acetobacterium pleiomorpha Actinoplanes tolypomycinaAtrhrobacter woodii Actibacter rectilineatus Anabaena protophormiaeAcetofilamentum Actibacter Actinoplanes Anabaena cylindrica ArthrobacterAcetofilamentum sediminis regularis Anabaena flosaquaepsychrolactophilus rigidum Actinoalloteichus Actinoplanes Anabaenavariabilis Arthrobacter Acetohalobium Actinoalloteichus teichomyceticusAnaeroarcus ramosus Acetohalobium cyanogriseus Actinoplanes AnaeroarcusArthrobacter arabaticum Actinoalloteichus utahensis burkinensissulfonivorans Acetomicrobium hymeniacidonis ActinopolysporaAnaerobaculum Arthrobacter Acetomicrobium ActinoalloteichusActinopolyspora Anaerobaculum sulfureus faecale spitiensis halophilamobile Arthrobacter Acetomicrobium Actinobaccillus ActinopolysporaAnaerobiospirillum uratoxydans flavidum Actinobacillus mortivallisAnaerobiospirillum Arthrobacter Acetonema capsulatus Actinosynnemasucciniciproducens ureafaciens Acetonema longum ActinobacillusActinosynnema Anaerobiospirillum Arthrobacter Acetothermus delphinicolamirum thomasii viscosus Acetothermus Actinobacillus ActinotaleaAnaerococcus Arthrobacter paucivorans hominis Actinotalea Anaerococcuswoluwensis Acholeplasma Actinobacillus fermentans hydrogenalis AsaiaAcholeplasma indolicus Aerococcus Anaerococcus Asaia bogorensis axanthumActinobacillus Aerococcus lactolyticus Asanoa Acholeplasma lignieresiisanguinicola Anaerococcus Asanoa ferruginea brassicae ActinobacillusAerococcus urinae prevotii Asticcacaulis Acholeplasma minor AerococcusAnaerococcus Asticcacaulis cavigenitalium Actinobacillus urinaeequitetradius biprosthecium Acholeplasma muris Aerococcus AnaerococcusAsticcacaulis equifetale Actinobacillus urinaehominis vaginalisexcentricus Acholeplasma pleuropneumoniae Aerococcus AnaerofustisAtopobacter granularum Actinobacillus viridans Anaerofustis AtopobacterAcholeplasma porcinus Aeromicrobium stercorihominis phocae hippikonActinobacillus Aeromicrobium Anaeromusa Atopobium Acholeplasma rossiierythreum Anaeromusa Atopobium fossor laidlawii Actinobacillus Aeromonasacidaminophila Atopobium Acholeplasma scotiae Aeromonas Anaeromyxobacterminutum modicum Actinobacillus allosaccharophila AnaeromyxobacterAtopobium Acholeplasma seminis Aeromonas dehalogenans parvulum morumActinobacillus bestiarum Anaerorhabdus Atopobium rimae Acholeplasmasuccinogenes Aeromonas caviae Anaerorhabdus Atopobium vaginaemultilocale Actinobaccillus Aeromonas furcosa AureobacteriumAcholeplasma suis encheleia Anaerosinus Aureobacterium oculiActinobacillus Aeromonas Anaerosinus barkeri Acholeplasma ureaeenteropelogenes glycerini Aurobacterium palmae Actinobaculum AeromonasAnaerovirgula Aurobacterium Acholeplasma Actinobaculum eucrenophilaAnaerovirgula liquefaciens parvum massiliense Aeromonas multivoransAvibacterium Acholeplasma Actinobaculum ichthiosmia AncalomicrobiumAvibacterium avium pleciae schaalii Aeromonas AncalomicrobiumAvibacterium Acholeplasma Actinobaculum jandaei adetum gallinarum vitulisuis Aeromonas media Ancylobacter Avibacterium Achromobacter ActinomycesAeromonas Ancylobacter paragallinarum Achromobacter urinale popoffiiaquaticus Avibacterium denitrificans Actinocatenispora Aeromonas sobriaAneurinibacillus volantium Achromobacter Actinocatenispora Aeromonasveronii Aneurinibacillus Azoarcus insolitus rupis Agrobacteriumaneurinilyticus Azoarcus indigens Achromobacter ActinocatenisporaAgrobacterium Aneurinibacillus Azoarcus piechaudii thailandicagelatinovorum migulanus tolulyticus Achromobacter ActinocatenisporaAgrococcus Aneurinibacillus Azoarcus ruhlandii sera Agrococcusthermoaerophilus toluvorans Achromobacter Actinocorallia citreusAngiococcus Azohydromonas spanius Actinocorallia Agrococcus AngiococcusAzohydromonas Acidaminobacter aurantiaca jenensis disciformis australicaAcidaminobacter Actinocorallia Agromonas Angulomicrobium Azohydromonashydrogenoformans aurea Agromonas Angulomicrobium lata AcidaminococcusActinocorallia oligotrophica tetraedrale Azomonas Acidaminococcuscavernae Agromyces Anoxybacillus Azomonas agilis fermentansActinocorallia Agromyces Anoxybacillus Azomonas insignis Acidaminococcusglomerata fucosus pushchinoensis Azomonas intestini ActinocoralliaAgromyces Aquabacterium macrocytogenes Acidicaldus herbida hippuratusAquabacterium Azorhizobium Acidicaldus Actinocorallia Agromyces communeAzorhizobium organivorans libanotica luteolus Aquabacterium caulinodansAcidimicrobium Actinocorallia Agromyces parvum AzorhizophilusAcidimicrobium longicatena mediolanus Azorhizophilus ferrooxidansActinomadura Agromyces paspali Acidiphilium Actinomadura alba ramosusAzospirillum Acidiphilium Actinomadura Agromyces Azospirillumacidophilum atramentaria rhizospherae brasilense AcidiphiliumActinomadura Akkermansia Azospirillum angustum bangladeshensisAkkermansia halopraeferens Acidiphilium Actinomadura muciniphilaAzospirillum cryptum catellatispora Albidiferax irakense AcidiphiliumActinomadura Albidiferax Azotobacter multivorum chibensis ferrireducensAzotobacter Acidiphilium Actinomadura Albidovulum beijerinckiiorganovorum chokoriensis Albidovulum Azotobacter AcidiphiliumActinomadura inexpectatum chroococcum rubrum citrea AlcaligenesAzotobacter Acidisoma Actinomadura Alcaligenes nigricans Acidisomacoerulea denitrificans Azotobacter sibiricum Actinomadura Alcaligenessalinestris Acidisoma tundrae echinospora faecalis AzotobacterAcidisphaera Actinomadura Alcanivorax vinelandii Acidisphaera fibrosaAlcanivorax rubrifaciens Actinomadura borkumensis Acidithiobacillusformosensis Alcanivorax Acidithiobacillus Actinomadura jadensisalbertensis hibisca Algicola Acidithiobacillus Actinomadura Algicolacaldus kijaniata bacteriolytica Acidithiobacillus ActinomaduraAlicyclobacillus ferrooxidans latina Alicyclobacillus AcidithiobacillusActinomadura disulfidooxidans thiooxidans livida AlicyclobacillusAcidobacterium Actinomadura sendaiensis Acidobacterium luteofluorescensAlicyclobacillus capsulatum Actinomadura vulcanalis macra AlishewanellaActinomadura Alishewanella madurae fetalis Actinomadura Alkalibacillusoligospora Alkalibacillus Actinomadura haloalkaliphilus pelletieriActinomadura rubrobrunea Actinomadura rugatobispora Actinomadura umbrinaActinomadura verrucosospora Actinomadura vinacea Actinomaduraviridilutea Actinomadura viridis Actinomadura yumaensis BacillusBacteroides Bibersteinia Borrelia Brevinema [see below] BacteroidesBibersteinia trehalosi Borrelia afzelii Brevinema Bacteriovorax caccaeBifidobacterium Borrelia americana andersonii Bacteriovorax BacteroidesBifidobacterium Borrelia Brevundimonas stolpii coagulans adolescentisburgdorferi Brevundimonas Bacteroides Bifidobacterium Borrelia albaeggerthii angulatum carolinensis Brevundimonas BacteroidesBifidobacterium Borrelia coriaceae aurantiaca fragilis animalis Borreliagarinii Brevundimonas Bacteroides Bifidobacterium Borrelia japonicadiminuta galacturonicus asteroides Bosea Brevundimonas BacteroidesBifidobacterium Bosea intermedia helcogenes bifidum minatitlanensisBrevundimonas Bacteroides Bifidobacterium boum Bosea thiooxidanssubvibrioides ovatus Bifidobacterium breve Brachybacterium BrevundimonasBacteroides Bifidobacterium Brachybacterium vancanneytii pectinophiluscatenulatum alimentarium Brevundimonas Bacteroides BifidobacteriumBrachybacterium variabilis pyogenes choerinum faecium BrevundimonasBacteroides Bifidobacterium Brachybacterium vesicularis salyersiaecoryneforme paraconglomeratum Brochothrix Bacteroides BifidobacteriumBrachybacterium Brochothrix stercoris cuniculi rhamnosum campestrisBacteroides suis Bifidobacterium Brachybacterium Brochothrix Bacteroidesdentium tyrofermentans thermosphacta tectus Bifidobacterium BrachyspiraBrucella Bacteroides gallicum Brachyspira Brucella canisthetaiotaomicron Bifidobacterium alvinipulli Brucella Bacteroidesgallinarum Brachyspira neotomae uniformis Bifidobacterium hyodysenteriaeBryobacter Bacteroides indicum Brachyspira Bryobacter ureolyticusBifidobacterium longum innocens aggregatus Bacteroides BifidobacteriumBrachyspira Burkholderia vulgatus magnumBifidobacterium murdochiiBurkholderia Balnearium merycicum Brachyspira ambifaria BalneariumBifidobacterium pilosicoli Burkholderia lithotrophicum minimumBradyrhizobium andropogonis Balneatrix Bifidobacterium BradyrhizobiumBurkholderia Balneatrix alpica pseudocatenulatum canariense anthinaBalneola Bifidobacterium Bradyrhizobium Burkholderia Balneola vulgarispseudolongum elkanii caledonica Barnesiella BifidobacteriumBradyrhizobium Burkholderia Barnesiella pullorum japonicum caryophylliviscericola Bifidobacterium Bradyrhizobium Burkholderia Bartonellaruminantium liaoningense cenocepacia Bartonella BifidobacteriumBrenneria Burkholderia alsatica saeculare Brenneria alni cepaciaBartonella Bifidobacterium subtile Brenneria Burkholderia bacilliformisBifidobacterium nigrifluens cocovenenans Bartonella thermophilumBrenneria quercina Burkholderia clarridgeiae Bilophila Brenneriaquercina dolosa Bartonella Bilophila wadsworthia Brenneria salicisBurkholderia doshiae Biostraticola Brevibacillus fungorum BartonellaBiostraticola tofi Brevibacillus agri Burkholderia elizabethae BizioniaBrevibacillus glathei Bartonella Bizionia argentinensis borstelensisBurkholderia grahamii Blastobacter Brevibacillus brevis glumaeBartonella Blastobacter capsulatus Brevibacillus Burkholderia henselaeBlastobacter centrosporus graminis Bartonella denitrificansBrevibacillus Burkholderia rochalimae Blastococcus choshinensiskururiensis Bartonella Blastococcus Brevibacillus Burkholderia vinsoniiaggregatus invocatus multivorans Bavariicoccus BlastococcusBrevibacillus Burkholderia Bavariicoccus saxobsidens laterosporusphenazinium seileri Blastochloris Brevibacillus BurkholderiaBdellovibrio Blastochloris parabrevis plantarii Bdellovibrio viridisBrevibacillus Burkholderia bacteriovorus Blastomonas reuszeri pyrrociniaBdellovibrio Blastomonas Brevibacterium Burkholderia exovorus natatoriaBrevibacterium silvatlantica Beggiatoa Blastopirellula abidumBurkholderia Beggiatoa alba Blastopirellula Brevibacterium stabilisBeijerinckia marina album Burkholderia Beijerinckia BlautiaBrevibacterium thailandensis derxii Blautia coccoides aurantiacumBurkholderia Beijerinckia Blautia hansenii Brevibacterium tropicafluminensis Blautia producta celere Burkholderia Beijerinckia Blautiawexlerae Brevibacterium unamae indica Bogoriella epidermidisBurkholderia Beijerinckia Bogoriella Brevibacterium vietnamiensismobilis caseilytica frigoritolerans Buttiauxella Belliella BordetellaBrevibacterium Buttiauxella Belliella baltica Bordetella aviumhalotolerans agrestis Bellilinea Bordetella Brevibacterium ButtiauxellaBellilinea bronchiseptica iodinum brennerae caldifistulae Bordetellahinzii Brevibacterium Buttiauxella Belnapia Bordetella holmesii linensferragutiae Belnapia Bordetella parapertussis BrevibacteriumButtiauxella moabensis Bordetella pertussis lyticum gaviniae BergeriellaBordetella petrii Brevibacterium Buttiauxella Bergeriella Bordetellatrematum mcbrellneri izardii denitrificans Brevibacterium ButtiauxellaBeutenbergia otitidis noackiae Beutenbergia Brevibacterium Buttiauxellacavernae oxydans warmboldiae Brevibacterium Butyrivibrio paucivoransButyrivibrio Brevibacterium fibrisolvens stationis Butyrivibrio hungateiButyrivibrio proteoclasticus Bacillus B. acidiceler B. aminovorans B.glucanolyticus B. taeanensis B. lautus B. acidicola B. amylolyticus B.gordonae B. tequilensis B. lehensis B. acidiproducens B. andreesenii B.gottheilii B. thermantarcticus B. lentimorbus B. acidocaldarius B.aneurinilyticus B. graminis B. thermoaerophilus B. lentus B.acidoterrestris B. anthracis B. halmapalus B. thermoamylovorans B.licheniformis B. aeolius B. aquimaris B. haloalkaliphilus B.thermocatenulatus B. ligniniphilus B. aerius B. arenosi B. halochares B.thermocloacae B. litoralis B. aerophilus B. arseniciselenatis B.halodenitrificans B. thermocopriae B. locisalis B. agaradhaerens B.arsenicus B. halodurans B. thermodenitrificans B. luciferensis B. agriB. aurantiacus B. halophilus B. thermoglucosidasius B. luteolus B.aidingensis B. arvi B. halosaccharovorans B. thermolactis B. luteus B.akibai B. aryabhattai B. hemicellulosilyticus B. thermoleovorans B.macauensis B. alcalophilus B. asahii B. hemicentroti B. thermophilus B.macerans B. algicola B. atrophaeus B. herbersteinensis B. thermoruber B.macquariensis B. alginolyticus B. axarquiensis B. horikoshii B.thermosphaericus B. macyae B. alkalidiazotrophicus B. azotofixans B.horneckiae B. thiaminolyticus B. malacitensis B. alkalinitrilicus B.azotoformans B. horti B. thioparans B. mannanilyticus B. alkalisediminisB. badius B. huizhouensis B. thuringiensis B. marisflavi B.alkalitelluris B. barbaricus B. humi B. tianshenii B. marismortui B.altitudinis B. bataviensis B. hwajinpoensis B. trypoxylicola B.marmarensis B. alveayuensis B. beijingensis B. idriensis B. tusciae B.massiliensis B. alvei B. benzoevorans B. indicus B. validus B.megaterium B. amyloliquefaciens B. beringensis B. infantis B.vallismortis B. mesonae B. a. subsp. amyloliquefaciens B. berkeleyi B.infernus B. vedderi B. methanolicus B. a. subsp. plantarum B. beveridgeiB. insolitus B. velezensis B. methylotrophicus B. dipsosauri B.bogoriensis B. invictae B. vietnamensis B. migulanus B. drentensis B.boroniphilus B. iranensis B. vireti B. mojavensis B. edaphicus B.borstelensis B. isabeliae B. vulcani B. mucilaginosus B. ehimensis B.brevis Migula B. isronensis B. wakoensis B. muralis B. eiseniae B.butanolivorans B. jeotgali B. weihenstephanensis B. murimartini B.enclensis B. canaveralius B. kaustophilus B. xiamenensis B. mycoides B.endophyticus B. carboniphilus B. kobensis B. xiaoxiensis B. naganoensisB. endoradicis B. cecembensis B. kochii B. zhanjiangensis B. nanhaiensisB. farraginis B. cellulosilyticus B. kokeshiiformis B. peoriae B.nanhaiisediminis B. fastidiosus B. centrosporus B. koreensis B.persepolensis B. nealsonii B. fengqiuensis B. cereus B. korlensis B.persicus B. neidei B. firmus B. chagannorensis B. kribbensis B. pervagusB. neizhouensis B. flexus B. chitinolyticus B. krulwichiae B.plakortidis B. niabensis B. foraminis B. chondroitinus B. laevolacticusB. pocheonensis B. niacini B. fordii B. choshinensis B. larvae B.polygoni B. novalis B. formosus B. chungangensis B. laterosporus B.polymyxa B. oceanisediminis B. fortis B. cibi B. salexigens B. popilliaeB. odysseyi B. fumarioli B. circulans B. saliphilus B. pseudalcalophilusB. okhensis B. funiculus B. clarkii B. schlegelii B. pseudofirmus B.okuhidensis B. fusiformis B. clausii B. sediminis B. pseudomycoides B.oleronius B. galactophilus B. coagulans B. selenatarsenatis B.psychrodurans B. oryzaecorticis B. galactosidilyticus B. coahuilensis B.selenitireducens B. psychrophilus B. oshimensis B. galliciensis B.cohnii B. seohaeanensis B. psychrosaccharolyticus B. pabuli B. gelatiniB. composti B. shacheensis B. psychrotolerans B. pakistanensis B.gibsonii B. curdlanolyticus B. shackletonii B. pulvifaciens B. pallidusB. ginsengi B. cycloheptanicus B. siamensis B. pumilus B. pallidus B.ginsengihumi B. cytotoxicus B. silvestris B. purgationiresistens B.panacisoli B. ginsengisoli B. daliensis B. simplex B. pycnus B.panaciterrae B. globisporus B. decisifrondis B. siralis B. qingdaonensisB. pantothenticus (eg, B. g. subsp. B. decolorationis B. smithii B.qingshengii B. parabrevis Globisporus; B. deserti B. soli B. reuszeri B.paraflexus or B. g. subsp. B. solimangrovi B. rhizosphaerae B. pasteuriiMarinus) B. solisalsi B. rigui B. patagoniensis B. songklensis B. rurisB. sonorensis B. safensis B. sphaericus B. salarius B. sporothermoduransB. stearothermophilus B. stratosphericus B. subterraneus B. subtilis(eg, B. s. subsp. Inaquosorum; or B. s. subsp. Spizizeni; or B. s.subsp. Subtilis) Caenimonas Campylobacter Cardiobacterium CatenuloplanesCurtobacterium Caenimonas koreensis Campylobacter coli CardiobacteriumCatenuloplanes Curtobacterium Caldalkalibacillus Campylobacter hominisatrovinosus albidum Caldalkalibacillus concisus CarnimonasCatenuloplanes Curtobacterium uzonensis Campylobacter Carnimonascastaneus citreus Caldanaerobacter curvus nigrificans CatenuloplanesCaldanaerobacter Campylobacter fetus Carnobacterium crispus subterraneusCampylobacter Carnobacterium Catenuloplanes Caldanaerobius gracilisalterfunditum indicus Caldanaerobius Campylobacter CarnobacteriumCatenuloplanes fijiensis helveticus divergens japonicus CaldanaerobiusCampylobacter Carnobacterium Catenuloplanes polysaccharolyticus hominisfunditum nepalensis Caldanaerobius zeae Campylobacter CarnobacteriumCatenuloplanes Caldanaerovirga hyointestinalis gallinarum nigerCaldanaerovirga Campylobacter Carnobacterium Chryseobacteriumacetigignens jejuni maltaromaticum Chryseobacterium CaldicellulosiruptorCampylobacter lari Carnobacterium balustinum CaldicellulosiruptorCampylobacter mobile Citrobacter bescii mucosalis Carnobacterium C.amalonaticus Caldicellulosiruptor Campylobacter viridans C. braakiikristjanssonii rectus Caryophanon C. diversus CaldicellulosiruptorCampylobacter Caryophanon C. farmeri owensensis showae latum C. freundiiCampylobacter Caryophanon C. gillenii sputorum tenue C. koseriCampylobacter Catellatospora C. murliniae upsaliensis Catellatospora C.pasteurii ^([1]) Capnocytophaga citrea C. rodentium CapnocytophagaCatellatospora C. sedlakii canimorsus methionotrophica C. werkmaniiCapnocytophaga Catenococcus C. youngae cynodegmi CatenococcusClostridium Capnocytophaga thiocycli (see below) gingivalis CoccochlorisCapnocytophaga Coccochloris granulosa elabens CapnocytophagaCorynebacterium haemolytica Corynebacterium Capnocytophaga flavescensochracea Corynebacterium Capnocytophaga variabile sputigena ClostridiumClostridium absonum, Clostridium aceticum, Clostridium acetireducens,Clostridium acetobutylicum, Clostridium acidisoli, Clostridiumaciditolerans, Clostridium acidurici, Clostridium aerotolerans,Clostridium aestuarii, Clostridium akagii, Clostridium aldenense,Clostridium aldrichii, Clostridium algidicarni, Clostridiumalgidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum,Clostridium alkalicellulosi, Clostridium aminophilum, Clostridiumaminovalericum, Clostridium amygdalinum, Clostridium amylolyticum,Clostridium arbusti, Clostridium arcticum, Clostridium argentinense,Clostridium asparagiforme, Clostridium aurantibutyricum, Clostridiumautoethanogenum, Clostridium baratii, Clostridium barkeri, Clostridiumbartlettii, Clostridium beijerinckii, Clostridium bifermentans,Clostridium bolteae, Clostridium bornimense, Clostridium botulinum,Clostridium bowmanii, Clostridium bryantii, Clostridium butyricum,Clostridium cadaveris, Clostridium caenicola, Clostridiumcaminithermale, Clostridium carboxidivorans, Clostridium carnis,Clostridium cavendishii, Clostridium celatum, Clostridiumcelerecrescens, Clostridium cellobioparum, Clostridiumcellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi,Clostridium cellulovorans, Clostridium chartatabidum, Clostridiumchauvoei, Clostridium chromiireducens, Clostridium citroniae,Clostridium clariflavum, Clostridium clostridioforme, Clostridiumcoccoides, Clostridium cochlearium, Clostridium colletant, Clostridiumcolicanis, Clostridium colinum, Clostridium collagenovorans, Clostridiumcylindrosporum, Clostridium difficile, Clostridium diolis, Clostridiumdisporicum, Clostridium drakei, Clostridium durum, Clostridiumestertheticum, Clostridium estertheticum estertheticum, Clostridiumestertheticum laramiense, Clostridium fallax, Clostridium felsineum,Clostridium fervidum, Clostridium fimetarium, Clostridiumformicaceticum, Clostridium frigidicarnis, Clostridium frigoris,Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii,Clostridium glycolicum, Clostridium glycyrrhizinilyticum, Clostridiumgrantii, Clostridium haemolyticum, Clostridium halophilum, Clostridiumhastiforme, Clostridium hathewayi, Clostridium herbivorans, Clostridiumhiranonis, Clostridium histolyticum, Clostridium homopropionicum,Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans,Clostridium hydroxybenzoicum, Clostridium hylemonae, Clostridiumjejuense, Clostridium indolis, Clostridium innocuum, Clostridiumintestinale, Clostridium irregulare, Clostridium isatidis, Clostridiumjosui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridiumlacusfryxellense, Clostridium laramiense, Clostridium lavalense,Clostridium lentocellum, Clostridium lentoputrescens, Clostridiumleptum, Clostridium limosum, Clostridium litorale, Clostridiumlituseburense, Clostridium ljungdahlii, Clostridium lortetii,Clostridium lundense, Clostridium magnum, Clostridium malenominatum,Clostridium mangenotii, Clostridium mayombei, Clostridiummethoxybenzovorans, Clostridium methylpentosum, Clostridiumneopropionicum, Clostridium nexile, Clostridium nitrophenolicum,Clostridium novyi, Clostridium oceanicum, Clostridium orbiscindens,Clostridium oroticum, Clostridium oxalicum, Clostridium papyrosolvens,Clostridium paradoxum, Clostridium paraperfringens (Alias: C. welchii),Clostridium paraputrificum, Clostridium pascui, Clostridiumpasteurianum, Clostridium peptidivorans, Clostridium perenne,Clostridium perfringens, Clostridium pfennigii, Clostridiumphytofermentans, Clostridium piliforme, Clostridium polysaccharolyticum,Clostridium populeti, Clostridium propionicum, Clostridiumproteoclasticum, Clostridium proteolyticum, Clostridium psychrophilum,Clostridium puniceum, Clostridium purinilyticum, Clostridiumputrefaciens, Clostridium putrificum, Clostridium quercicolum,Clostridium quinii, Clostridium ramosum, Clostridium rectum, Clostridiumroseum, Clostridium saccharobutylicum, Clostridium saccharogumia,Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum,Clostridium sardiniense, Clostridium sartagoforme, Clostridiumscatologenes, Clostridium schirmacherense, Clostridium scindens,Clostridium septicum, Clostridium sordellii, Clostridium sphenoides,Clostridium spiroforme, Clostridium sporogenes, Clostridiumsporosphaeroides, Clostridium stercorarium, Clostridium stercorariumleptospartum, Clostridium stercorarium stercorarium, Clostridiumstercorarium thermolacticum, Clostridium sticklandii, Clostridiumstraminisolvens, Clostridium subterminale, Clostridium sufflavum,Clostridium sulfidigenes, Clostridium symbiosum, Clostridium tagluense,Clostridium tepidiprofundi, Clostridium termitidis, Clostridium tertium,Clostridium tetani, Clostridium tetanomorphum, Clostridiumthermaceticum, Clostridium thermautotrophicum, Clostridiumthermoalcaliphilum, Clostridium thermobutyricum, Clostridiumthermocellum, Clostridium thermocopriae, Clostridiumthermohydrosulfuricum, Clostridium thermolacticum, Clostridiumthermopalmarium, Clostridium thermopapyrolyticum, Clostridiumthermosaccharolyticum, Clostridium thermosuccinogenes, Clostridiumthermosulfurigenes, Clostridium thiosulfatireducens, Clostridiumtyrobutyricum, Clostridium uliginosum, Clostridium ultunense,Clostridium villosum, Clostridium vincentii, Clostridium viride,Clostridium xylanolyticum, Clostridium xylanovorans DactylosporangiumDeinococcus Delftia Echinicola Dactylosporangium Deinococcus DelftiaEchinicola aurantiacum aerius acidovorans pacifica DactylosporangiumDeinococcus Desulfovibrio Echinicola fulvum apachensis Desulfovibriovietnamensis Dactylosporangium Deinococcus desulfuricans matsuzakienseaquaticus Diplococcus Dactylosporangium Deinococcus Diplococcus roseumaquatilis pneumoniae Dactylosporangium Deinococcus caeni thailandenseDeinococcus Dactylosporangium radiodurans vinaceum Deinococcusradiophilus Enterobacter Enterobacter kobei FaecalibacteriumFlavobacterium E. aerogenes E. ludwigii Faecalibacterium FlavobacteriumE. amnigenus E. mori prausnitzii antarcticum E. agglomerans E.nimipressuralis Fangia Flavobacterium E. arachidis E. oryzae Fangiaaquatile E. asburiae E. pulveris hongkongensis Flavobacterium E.cancerogenous E. pyrinus Fastidiosipila aquidurense E. cloacae E.radicincitans Fastidiosipila Flavobacterium E. cowanii E. tayloraesanguinis balustinum E. dissolvens E. turicensis FusobacteriumFlavobacterium E. gergoviae E. sakazakii Fusobacterium croceum E.helveticus Enterobacter soli nucleatum Flavobacterium E. hormaecheiEnterococcus cucumis E. intermedius Enterococcus Flavobacterium duransdaejeonense Enterococcus Flavobacterium faecalis defluvii EnterococcusFlavobacterium faecium degerlachei Erwinia Flavobacterium Erwiniahapontici denitrificans Escherichia Flavobacterium Escherichia colifilum Flavobacterium flevense Flavobacterium frigidarium Flavobacteriummizutaii Flavobacterium okeanokoites Gaetbulibacter HaemophilusIdeonella Janibacter Gaetbulibacter Haemophilus Ideonella Janibactersaemankumensis aegyptius azotifigens anophelis GallibacteriumHaemophilus Idiomarina Janibacter Gallibacterium anatis aphrophilusIdiomarina corallicola Gallicola Haemophilus felis abyssalis JanibacterGallicola barnesae Haemophilus Idiomarina limosus Garciella gallinariimbaltica Janibacter Garciella Haemophilus Idiomarina melonisnitratireducens haemolyticus fontislapidosi Janibacter GeobacillusHaemophilus Idiomarina terrae Geobacillus influenzae loihiensisJannaschia thermoglucosidasius Haemophilus Idiomarina JannaschiaGeobacillus paracuniculus ramblicola cystaugens stearothermophilusHaemophilus Idiomarina Jannaschia Geobacter parahaemolyticus seosinensishelgolandensis Geobacter Haemophilus Idiomarina Jannaschia bemidjiensisparainfluenzae zobellii pohangensis Geobacter brememis HaemophilusIgnatzschineria Jannaschia Geobacter chapellei paraphrohaemolyticusIgnatzschineria rubra Geobacter grbiciae Haemophilus larvaeJanthinobacterium Geobacter parasuis Ignavigranum Janthinobacteriumhydrogenophilus Haemophilus Ignavigranum agaricidamnosum Geobacterlovleyi pittmaniae ruoffiae Janthinobacterium Geobacter HafniaIlumatobacter lividum metallireducens Hafnia alvei Ilumatobacter JejuiaGeobacter pelophilus Hahella fluminis Jejuia Geobacter pickeringiiHahella Ilyobacter pallidilutea Geobacter ganghwensis IlyobacterJeotgalibacillus sulfurreducens Halalkalibacillus delafieldiiJeotgalibacillus Geodermatophilus Halalkalibacillus Ilyobacteralimentarius Geodermatophilus halophilus insuetus Jeotgalicoccusobscurus Helicobacter Ilyobacter Jeotgalicoccus GluconacetobacterHelicobacter polytropus halotolerans Gluconacetobacter pylori Ilyobacterxylinus tartaricus Gordonia Gordonia rubripertincta Kaistia LabedellaListeria ivanovii Micrococcus Nesterenkonia Kaistia adipata Labedella L.marthii Micrococcus Nesterenkonia Kaistia soli gwakjiensis L.monocytogenes luteus holobia Kangiella Labrenzia L. newyorkensisMicrococcus Nocardia Kangiella Labrenzia L. riparia lylae Nocardiaaquimarina aggregata L. rocourtiae Moraxella argentinensis Kangiellakoreensis Labrenzia alba L. seeligeri Moraxella bovis NocardiaKerstersia Labrenzia L. weihenstephanensis Moraxella corallinaKerstersia gyiorum alexandrii L. welshimeri nonliquefaciens NocardiaKiloniella Labrenzia marina Listonella Moraxella otitidiscaviarumKiloniella laminariae Labrys Listonella osloensis Klebsiella Labrysanguillarum Nakamurella K. granulomatis methylaminiphilus MacrococcusNakamurella K. oxytoca Labrys Macrococcus multipartita K. pneumoniaemiyagiensis bovicus Nannocystis K. terrigena Labrys monachusMarinobacter Nannocystis K. variicola Labrys Marinobacter pusillaKluyvera okinawensis algicola Natranaerobius Kluyvera ascorbata LabrysMarinobacter Natranaerobius Kocuria portucalensis bryozoorumthermophilus Kocuria roasea Lactobacillus Marinobacter NatranaerobiusKocuria varians [see below] flavimaris trueperi Kurthia LaceyellaMeiothermus Naxibacter Kurthia zopfii Laceyella putida MeiothermusNaxibacter Lechevalieria ruber alkalitolerans LechevalieriaMethylophilus Neisseria aerocolonigenes Methylophilus Neisseria cinereaLegionella methylotrophus Neisseria [see below] Microbacteriumdenitrificans Listeria Microbacterium Neisseria L. aquaticaammoniaphilum gonorrhoeae L. booriae Microbacterium Neisseria L.cornellensis arborescens lactamica L. fleischmannii MicrobacteriumNeisseria mucosa L. floridensis liquefaciens Neisseria sicca L.grandensis Microbacterium Neisseria subflava L. grayi oxydansNeptunomonas L. innocua Neptunomonas japonica Lactobacillus L.acetotolerans L. catenaformis L. mali L. parakefiri L. sakei L.acidifarinae L. ceti L. manihotivorans L. paralimentarius L. salivariusL. acidipiscis L. coleohominis L. mindensis L. paraplantarum L.sanfranciscensis L. acidophilus L. collinoides L. mucosae L. pentosus L.satsumensis Lactobacillus agilis L. composti L. murinus L. perolens L.secaliphilus L. algidus L. concavus L. nagelii L. plantarum L. sharpeaeL. alimentarius L. coryniformis L. namurensis L. pontis L. siliginis L.amylolyticus L. crispatus L. nantensis L. protectus L. spicheri L.amylophilus L. crustorum L. oligofermentans L. psittaci L. suebicus L.amylotrophicus L. curvatus L. oris L. rennini L. thailandensis L.amylovorus L. delbrueckii L. panis L. reuteri L. ultunensis L. animalissubsp. bulgaricus L. pantheris L. rhamnosus L. vaccinostercus L. antriL. delbrueckii L. parabrevis L. rimae L. vaginalis L. apodemi subsp.delbrueckii L. parabuchneri L. rogosae L. versmoldensis L. aviarius L.delbrueckii L. paracasei L. rossiae L. vini L. bifermentans subsp.lactis L. paracollinoides L. ruminis L. vitulinus L. brevis L.dextrinicus L. parafarraginis L. saerimneri L. zeae L. buchneri L.diolivorans L. homohiochii L. jensenii L. zymae L. camelliae L. equi L.iners L. johnsonii L. gastricus L. casei L. equigenerosi L. ingluviei L.kalixensis L. ghanensis L. kitasatonis L. farraginis L. intestinalis L.kefiranofaciens L. graminis L. kunkeei L. farciminis L. fuchuensis L.kefiri L. hammesii L. leichmannii L. fermentum L. gallinarum L. kimchiiL. hamsteri L. lindneri L. fornicalis L. gasseri L. helveticus L.harbinensis L. malefermentans L. fructivorans L. hilgardii L.hayakitensis L. frumenti Legionella Legionella Legionella CandidatusLegionella Legionella adelaidensis drancourtii jeonii quinlivaniiLegionella anisa Legionella Legionella Legionella Legionelladresdenensis jordanis rowbothamii beliardensis Legionella LegionellaLegionella Legionella drozanskii lansingensis rubrilucensbirminghamensis Legionella Legionella Legionella Legionella dumoffiilondiniensis sainthelensi bozemanae Legionella erythra LegionellaLegionella Legionella brunensis Legionella longbeachae santicrucisLegionella fairfieldensis Legionella lytica Legionella busanensisLegionella fallonii Legionella shakespearei Legionella cardiacaLegionella feeleii maceachernii Legionella Legionella cherrii LegionellaLegionella spiritensis Legionella geestiana massiliensis Legionellacincinnatiensis Legionella Legionella steelei Legionella genomospeciesmicdadei Legionella clemsonensis Legionella Legionella steigerwaltiiLegionella gormanii monrovica Legionella donaldsonii LegionellaLegionella taurinensis gratiana moravica Legionella LegionellaLegionella tucsonensis gresilensis nagasakiensis Legionella LegionellaLegionella tunisiensis hackeliae nautarum Legionella LegionellaLegionella wadsworthii impletisoli norrlandica Legionella LegionellaLegionella waltersii israelensis oakridgensis Legionella LegionellaLegionella worsleiensis jamestowniensis parisiensis LegionellaLegionella yabuuchiae pittsburghensis Legionella pneumophila Legionellaquateirensis Oceanibulbus Paenibacillus Prevotella QuadrisphaeraOceanibulbus Paenibacillus Prevotella Quadrisphaera indolifexthiaminolyticus albensis granulorum Oceanicaulis Pantoea Prevotellaamnii Quatrionicoccus Oceanicaulis Pantoea Prevotella Quatrionicoccusalexandrii agglomerans bergensis australiensis Oceanicola ParacoccusPrevotella bivia Quinella Oceanicola batsensis Paracoccus Prevotellabrevis Quinella ovalis Oceanicola alcaliphilus Prevotella Ralstoniagranulosus Paucimonas bryantii Ralstonia Oceanicola PaucimonasPrevotella buccae eutropha nanhaiensis lemoignei Prevotella RalstoniaOceanimonas Pectobacterium buccalis insidiosa Oceanimonas PectobacteriumPrevotella copri Ralstonia baumannii aroidearum Prevotellamannitolilytica Oceaniserpentilla Pectobacterium dentalis RalstoniaOceaniserpentilla atrosepticum Prevotella pickettii haliotisPectobacterium denticola Ralstonia Oceanisphaera betavasculorumPrevotella disiens pseudosolanacearum Oceanisphaera PectobacteriumPrevotella Ralstonia syzygii donghaensis cacticida histicola RalstoniaOceanisphaera Pectobacterium Prevotella solanacearum litoraliscarnegieana intermedia Ramlibacter Oceanithermus PectobacteriumPrevotella Ramlibacter Oceanithermus carotovorum maculosa henchirensisdesulfurans Pectobacterium Prevotella Ramlibacter Oceanithermuschrysanthemi marshii tataouinensis profundus Pectobacterium PrevotellaRaoultella Oceanobacillus cypripedii melaninogenica RaoultellaOceanobacillus caeni Pectobacterium Prevotella micans ornithinolyticaOceanospirillum rhapontici Prevotella Raoultella OceanospirillumPectobacterium multiformis planticola linum wasabiae PrevotellaRaoultella Planococcus nigrescens terrigena Planococcus Prevotellaoralis Rathayibacter citreus Prevotella oris RathayibacterPlanomicrobium Prevotella caricis Planomicrobium oulorum Rathayibacterokeanokoites Prevotella pallens festucae Plesiomonas Prevotella salivaeRathayibacter Plesiomonas Prevotella iranicus shigelloides stercoreaRathayibacter Proteus Prevotella rathayi Proteus vulgaris tanneraeRathayibacter Prevotella toxicus timonensis Rathayibacter Prevotellatritici veroralis Rhodobacter Providencia Rhodobacter Providenciasphaeroides stuartii Ruegeria Pseudomonas Ruegeria Pseudomonasgelatinovorans aeruginosa Pseudomonas alcaligenes Pseudomonasanguillispetica Pseudomonas fluorescens Pseudoalteromonas haloplanktisPseudomonas mendocina Pseudomonas pseudoalcaligenes Pseudomonas putidaPseudomonas tutzeri Pseudomonas syringae Psychrobacter Psychrobacterfaecalis Psychrobacter phenylpyruvicus Saccharococcus SagittulaSanguibacter Stenotrophomonas Tatlockia Saccharococcus Sagittulastellata Sanguibacter Stenotrophomonas Tatlockia thermophilusSalegentibacter keddieii maltophilia maceachernii SaccharomonosporaSalegentibacter Sanguibacter Streptococcus Tatlockia Saccharomonosporasalegens suarezii [also see below] micdadei azurea SalimicrobiumSaprospira Streptomyces Tenacibaculum Saccharomonospora SalimicrobiumSaprospira Streptomyces Tenacibaculum cyanea album grandis achromogenesamylolyticum Saccharomonospora Salinibacter Sarcina StreptomycesTenacibaculum viridis Salinibacter ruber Sarcina maxima cesalbusdiscolor Saccharophagus Salinicoccus Sarcina ventriculi StreptomycesTenacibaculum Saccharophagus Salinicoccus Sebaldella cescaepitosusgallaicum degradans alkaliphilus Sebaldella Streptomyces TenacibaculumSaccharopolyspora Salinicoccus termitidis cesdiastaticus lutimarisSaccharopolyspora hispanicus Serratia Streptomyces Tenacibaculumerythraea Salinicoccus roseus Serratia fonticola cesexfoliatusmesophilum Saccharopolyspora Salinispora Serratia StreptomycesTenacibaculum gregorii Salinispora marcescens fimbriatus skagerrakenseSaccharopolyspora arenicola Sphaerotilus Streptomyces Tepidanaerobacterhirsuta Salinispora tropica Sphaerotilus fradiae TepidanaerobacterSaccharopolyspora Salinivibrio natans Streptomyces syntrophicus hordeiSalinivibrio Sphingobacterium fulvissimus Tepidibacter Saccharopolysporacosticola Sphingobacterium Streptomyces Tepidibacter rectivirgulaSalmonella multivorum griseoruber formicigenes SaccharopolysporaSalmonella bongori Staphylococcus Streptomyces Tepidibacter spinosaSalmonella enterica [see below] griseus thalassicus SaccharopolysporaSalmonella Streptomyces Thermus taberi subterranea lavendulae ThermusSaccharothrix Salmonella typhi Streptomyces aquaticus Saccharothrixphaeochromogenes Thermus australiensis Streptomyces filiformisSaccharothrix thermodiastaticus Thermus coeruleofusca Streptomycesthermophilus Saccharothrix tubercidicus espanaensis Saccharothrixlongispora Saccharothrix mutabilis Saccharothrix syringae Saccharothrixtangerinus Saccharothrix texasensis Staphylococcus S. arlettae S.equorum S. microti S. schleiferi S. agnetis S. felis S. muscae S. sciuriS. aureus S. fleurettii S. nepalensis S. simiae S. auricularis S.gallinarum S. pasteuri S. simulans S. capitis S. haemolyticus S.petrasii S. stepanovicii S. caprae S. hominis S. pettenkoferi S.succinus S. carnosus S. hyicus S. piscifermentans S. vitulinus S.caseolyticus S. intermedius S. pseudintermedius S. warneri S.chromogenes S. kloosii S. pseudolugdunensis S. xylosus S. cohnii S. leeiS. pulvereri S. condimenti S. lentus S. rostri S. delphini S.lugdunensis S. saccharolyticus S. devriesei S. lutrae S. saprophyticusS. epidermidis S. lyticans S. massiliensis Streptococcus StreptococcusStreptococcus Streptococcus Streptococcus agalactiae infantariusorisratti thermophilus Streptococcus anginosus Streptococcus iniaeStreptococcus Streptococcus Streptococcus bovis Streptococcusparasanguinis sanguinis Streptococcus canis intermedius StreptococcusStreptococcus Streptococcus Streptococcus peroris sobrinus constellatuslactarius Streptococcus Streptococcus Streptococcus downei Streptococcuspneumoniae suis Streptococcus milleri Streptococcus Streptococcusdysgalactiae Streptococcus mitis pseudopneumoniae uberis Streptococcusequines Streptococcus Streptococcus Streptococcus Streptococcus faecalismutans pyogenes vestibularis Streptococcus ferus Streptococcus oralisStreptococcus Streptococcus Streptococcus ratti viridans tigurinusStreptococcus Streptococcus salivariu zooepidemicus UliginosibacteriumVagococcus Vibrio Virgibacillus Xanthobacter UliginosibacteriumVagococcus Vibrio aerogenes Virgibacillus Xanthobacter gangwonensecarniphilus Vibrio halodenitrificans agilis Ulvibacter Vagococcusaestuarianus Virgibacillus Xanthobacter Ulvibacter litoralis elongatusVibrio albensis pantothenticus aminoxidans Umezawaea Vagococcus fessusVibrio Weissella Xanthobacter Umezawaea tangerina Vagococcus fluvialisalginolyticus Weissella cibaria autotrophicus Undibacterium Vagococcuslutrae Vibrio campbellii Weissella confusa Xanthobacter Undibacteriumpigrum Vagococcus Vibrio cholerae Weissella flavus Ureaplasmasalmoninarum Vibrio halotolerans Xanthobacter Ureaplasma Variovoraxcincinnatiensis Weissella tagetidis urealyticum Variovorax Vibriohellenica Xanthobacter Ureibacillus boronicumulans coralliilyticusWeissella viscosus Ureibacillus composti Variovorax Vibrio kandleriXanthomonas Ureibacillus dokdonensis cyclitrophicus WeissellaXanthomonas suwonensis Variovorax Vibrio koreensis albilineansUreibacillus terrenus paradoxus diazotrophicus Weissella minorXanthomonas Ureibacillus Variovorax soli Vibrio fluvialis Weissellaalfalfae thermophilus Veillonella Vibrio furnissii paramesenteroidesXanthomonas Ureibacillus Veillonella atypica Vibrio gazogenes Weissellasoli arboricola thermosphaericus Veillonella caviae Vibrio halioticoliWeissella Xanthomonas Veillonella criceti Vibrio harveyi thailandensisaxonopodis Veillonella dispar Vibrio Weissella Xanthomonas Veillonellaichthyoenteri viridescens campestris montpellierensis Vibrio WilliamsiaXanthomonas Veillonella parvula mediterranei Williamsia citriVeillonella ratti Vibrio marianensis Xanthomonas Veillonellametschnikovii Williamsia maris codiaei rodentium Vibrio mytiliWilliamsia Xanthomonas Venenivibrio Vibrio natriegens serinedenscucurbitae Venenivibrio Vibrio Winogradskyella Xanthomonasstagnispumantis navarrensis Winogradskyella euvesicatoriaVerminephrobacter Vibrio nereis thalassocola XanthomonasVerminephrobacter Vibrio Wolbachia fragariae eiseniae nigripulchritudoWolbachia Xanthomonas Verrucomicrobium Vibrio ordalii persica fuscansVerrucomicrobium Vibrio orientalis Wolinella Xanthomonas spinosum VibrioWolinella gardneri parahaemolyticus succinogenes Xanthomonas Vibriopectenicida Zobellia hortorum Vibrio penaeicida Zobellia XanthomonasVibrio galactanivorans hyacinthi proteolyticus Zobellia uliginosaXanthomonas Vibrio shilonii Zoogloea perforans Vibrio splendidusZoogloea Xanthomonas Vibrio tubiashii ramigera phaseoli Vibriovulnificus Zoogloea Xanthomonas resiniphila pisi Xanthomonas populiXanthomonas theicola Xanthomonas translucens Xanthomonas vesicatoriaXylella Xylella fastidiosa Xylophilus Xylophilus ampelinus XenophilusYangia Yersinia Zooshikella Zobellella Xenophilus azovorans Yangiapacifica mollaretii Zooshikella Zobellella Xenorhabdus Yaniella Yersiniaganghwensis denitrificans Xenorhabdus beddingii Yaniella flavaphilomiragia Zunongwangia Zobellella Xenorhabdus bovienii YaniellaYersinia pestis Zunongwangia taiwanensis Xenorhabdus halotoleransYersinia profunda Zeaxanthinibacter cabanillasii Yeosuanapseudotuberculosis Zymobacter Zeaxanthinibacter Xenorhabdus doucetiaeYeosuana Yersinia rohdei Zymobacter enoshimensis Xenorhabdus griffiniaearomativorans Yersinia ruckeri palmae Zhihengliuella Xenorhabdushominickii Yersinia Yokenella Zymomonas Zhihengliuella XenorhabdusYersinia aldovae Yokenella Zymomonas halotolerans koppenhoeferi Yersiniabercovieri regensburgei mobilis Xylanibacterium Xenorhabdus YersiniaYonghaparkia Zymophilus Xylanibacterium nematophila enterocoliticaYonghaparkia Zymophilus ulmi Xenorhabdus poinarii Yersinia alkaliphilapaucivorans Xylanibacter entomophaga Zavarzinia Zymophilus Xylanibacteroryzae Yersinia Zavarzinia raffinosivorans frederiksenii compransorisYersinia intermedia Yersinia kristensenii

TABLE 2 Systemic inflammatory response syndrome Finding ValueTemperature <36° C. (96.8° F.) or >38° C. (100.4° F.) Heart rate >90/minRespiratory rate >20/min or PaCO2 < 32 mmHg (4.3 kPa) WBC <4 × 10⁹/L(<4000/mm³), >12 × 10⁹/L (>12,000/mm³), or 10% bands

TABLE 3 Sequences of genomic targets to selectivelyCRISPR-kill E. coli strains. The 3-bp PAM are shown in bold type (ie, tgg, aggand cgg). Expected SpCas9-gRNA cleavagesites are indicated by vertical arrows (3 bp upstream of the PAM). Target Target strain gene Genomic target sequence E. coli 23S

(EHEC) ribosomal (SEQ ID NO: 1) ATCC43888 RNA E. coli yapH                

Nissle cacccgcattacctttatgcagg 1917 (SEQ ID NO: 2) E. coli pks                

Nisslte gcgcgggtgtggttgtgcttcgg 1917 (SEQ ID NO: 3)

TABLE 4 Sequences of aenomic targets & Cas toselectively CRISPR-kill C dificile(a) Overview of design spacers in pMTL84151 -cdCRISPR1 targeting C. difficile Gene Name PAM target tcdA TA2 CCAACACCTTAACCCAGCCATAGAGTCTGATAA TAACTTC (SEQ ID NO: 4) tcdB TB2 CCAGACTTATTTGAGTCTATAGAGAAACCTAGT TCAGTAA (SEQ ID NO: 5) rRNA-1 TRR1 CCTTCGACGACTTCTTCCAAAAGGTTAGATAAT CGGCTTC (SEQ ID NO: 6) rRNA-2 TRR2 CCAGTACAGGATGGACCCGCGTCTGATTAGCTA GTTGGTA (SEQ ID NO: 7) gyrA TAR1 CCATCCTCATGGAGATACTGCTGTTTATTATGC TATGGTA (SEQ ID NO: 8)(b) Cas3 sequence (SEQ ID NO: 9) SequenceTCAAATAAATTGGTCTATTTCATTACTTATAAGCACACCTTTACCAAATATCTGTTTTGTATGCTTATTTTCGTATATATCATATTTATATAAAAGTATTTTTAAATCTTCAAGACCTTTCACCTGTATGTCAATTACATTTTGTTTAGCTTTATATATTGGTAAATTTACAGTTTTTTTAATTATCTCTCTTCTTGCTTTTTTTCTCTTAGACTTTATTTTATTTATCAATTGTCTATCATCATTACTATATGCATTTATAAGCTCTTGTCCCAACTCTTCATAATCTTTGATTAAAGTTTCTTCAATTTTATTATATATATCCTCTGGAATTACTGTATATCCTTCAATATTTCTAAGTATATTTTGTGCATCTTTAGAACCTAAACTATATGGCGTTATAGTATCTAAAATATTTAAAGCACTGGTAAATCTCTTTTCAAAAGCTGTACCTTCTAGACTTTCTTTAGAGTATAGTATCTTAACCATTTCTACCTTATATTTTTCTTTCATCTTCACACATTCTTTTCCATTTATAAAAGTTTTCAGTAGCTCAAGTCCTTTTTCTACAATATCCTTATCATAAATACTACCTATTCCTGTAGCTTTTTCTGTATATATAAATATATTTGGGCTATTTTCTTCATACTCACGACTTCTATAACATCTACCAAATCGTTGAAATAGACTGTCAAGTGTTGAATTTTCTGTATGAAGCTCATCAAAATCAATATCAAGGGATGCTTCCACTAATTGTGTAGTAATCCAAATACCATTACTATCACTATCTGCAAATTCTTTTATATATTTTTCTAATTTTGCTCTATCTTCTTGTATATACATAGAATGTAAAAGATTTAAGTTGACATCTATTCCTTTTGGCTTAACTATTTCTTCTATTAACTCATATTTTTCTACAGCACTTTTAACTGTATTTACTATTACAAGAACTTTTTTATTCATTCCACTTTGTATTATTTTACCTAAGTTTTCATCTATTGAATTTTCTACAATAGACACACAATGTCTTATTTTTTCTGTATTACATGTCAATTCAGCTAAGTTACTATTCATTACACCTCTTTTTTTTAATTCATCTATATATATGGTTGGCATAGTAGCTGTCATTATCATAAATCTGCCACCTATCTTATGTATCATTTCTATACCTTTTACCAATACAGCTGCTATTTCTGGTGAATATGCTTGTATCTCATCTATTACTACTTTTGAATATGCTAATGTTGAGTACACTTTTTCATACCCTCTATACAAAAAAGGAAATTTAAATATTTGGTCTATTGTAGAAAATGTCAGTTTGCAAGATAATAACTTTGCTAAATCTACAATCTCACTTGAATTTTCTTGATTACTTTCTTCTAGATAATCTATTGCTGTTGAATGTAACAAACCTAAGAATGTATCATTTGATTCTCCAACTCCAACTATATTTTTTGCTCTATCAAATAATGCATTTATACTTACTCTTAATGGAAGTGTAAAAAATGCCTTGTCTTTATCTATCCAAATTAAGGCAGTTTCTGTTTTTCCCATTCCTGTAGGTGCAATCAGTATTATATTCTTATTTCTATTAGATTTAGCAAATGATTGAGCCTCTCTCAAACTACCAAACTCTTTCATTAAATAATTTTCTGTCTGTTCTCCTATATTTATAACATTATTGCATTCCACAACTTCATGAGCAGAAGCACTGTGGTCTAATCTATGTAGTATTCCTTTTAACATAATATATAAATTATAGTACTTGTGATTTTTATCTATTCTTTTTTCTACACTTTGTAGATATACTTTACTCAATTTTTCTGTTTTTATTGGATATCTAACTTTAAATTCATGCTGTAGTTCATAAACTTTATTTATTAAATCTTCATCTAATATTTTTTGTATTAAATTTTTAAAATCTTTATCTATAAAGATATCTCTTTCATGATGATATACAATAACTTGATTTAATACTGCTCTAAGTTCTTTATTTTTTTTCCTGTCTATATAACTATAATCAATAAATGCAGGAGAAAGATAATTATGGCCTACATTATTTTCTAAATGAGTTACTATTTTAGGTTCATTTATTTTAGTATCCATTCTGCTTTTTATTAACTCTTGAAACGGTGAAAATGCTTTTCCAATATCATGAAATTCTATAACAAAGTCAAGTAATTGCCAAAATATCTCCTCTTCTAGAAAATCTAAGCTATTTATATTTTTTCCATAACTTTCTCTTAATACATTCATTTGTTTTAAAAGTTCATCAGTATGTTCTCTAAGTGTTTCCACTGGATTAGATTTAGCATATAACAT

TABLE 4 Sequences of Cas9 used to selectively kill E coli.SEQ ID NO: 10 (Cas9 nucleotide sequence)ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGG TGACTGASEQ ID NO: 11 (Cas9 amino acid sequence)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD

TABLE 5 E. coli/K. pneumoniae/ Gram-neg. bacteria P. aeruginosa ascausative as causative pathogens in bacteraemia infectious pathogen incancer patients Publication in cancer patients [fraction of Gram-neg.cases] Samonis et al 65% 54% [85%] Velasco et al 45% 33% [74%] Marín etal 55% 51% [92%] Anatoliotaki et al 47% 34% [73%]

What is claimed is:
 1. A method of treating or reducing the risk of aurinary tract infection caused by first bacteria in a subject, themethod comprising: selectively killing the first bacteria in the subjectby cutting a target site comprised by the genomes of the first bacteriausing an RNA-guided Cas nuclease, wherein the method comprisesadministering to the subject a nucleic acid sequence comprising orencoding one or more guide RNAs (gRNAs) at a first time (T1) and at asecond time (T2), wherein T2 is at least 3 hours after T1, wherein theone or more gRNAs hybridize to the target site to guide the Cas nucleaseto cut the target site, thereby killing the first bacteria, wherein thesubject comprises second bacteria of one or more strains or species thatare different from that of the first bacteria, wherein the genomes ofthe second bacteria do not comprise the target site, wherein the genomesof the second bacteria are not cut by the Cas nuclease in the subject,whereby second bacteria survive in the presence of the Cas nuclease inthe subject, and wherein the number of the first bacteria is reduced atleast 100-fold by the first 30 minutes after exposing the subject to theone or more gRNAs.
 2. The method of claim 1, wherein the number of thefirst bacteria is reduced at least 1000-fold by the first 30 minutesafter exposing the subject to the one or more gRNAs.
 3. The method ofclaim 1, wherein the number of the first bacteria is reduced at least100-fold by the first 15 minutes after exposing the subject to the oneor more gRNAs.
 4. The method of claim 1, wherein the number of the firstbacteria is reduced at least 1000-fold by the first 15 minutes afterexposing the subject to the one or more gRNAs.
 5. The method of claim 1,wherein the reduction in the number of the first bacteria persists forat least 30 minutes immediately after the first 30 minutes afterexposing the subject to the one or more gRNAs.
 6. The method of claim 2,wherein the reduction in the number of the first bacteria by at least100-fold is maintained for at least 120 minutes after exposing thesubject to the one or more gRNAs.
 7. The method of claim 1, wherein thegRNA comprises a sequence capable of hybridizing to a protospacersequence comprising the target site, wherein the protospacer sequence is15-45 nucleotides in length.
 8. The method of claim 7, wherein theprotospacer sequence is 15-25 nucleotides in length.
 9. The method ofclaim 1, wherein the target site is in a protospacer sequence that isadjacent to an NGG, NAG, NGA, NGC, NGGNG, NNGRRT or NNAGAAW protospaceradjacent motif (PAM).
 10. The method of claim 1, wherein the nucleicacid sequence is or is comprised by a vector.
 11. The method of claim10, wherein the vector is a phage, phagemid, viriophage, virus, plasmid,or transposon.
 12. The method of claim 10, wherein the vector is aconjugative plasmid.
 13. The method of claim 12, wherein the conjugativeplasmid is delivered from carrier bacteria.
 14. The method of claim 10,comprising A method of selectively killing first bacteria in a subject,the method comprising: selectively killing the first bacteria in thesubject by cutting a target site comprised by the genomes of the firstbacteria using an RNA-guided Cas nuclease, wherein the method comprisesadministering to the subject a nucleic acid sequence comprising orencoding one or more guide RNAs (gRNAs), wherein the nucleic acidsequence is or is comprised by a first nucleic acid vector, and whereinthe method comprises administering a second nucleic acid vector to thesubject, wherein the second vector encodes the Cas nuclease, wherein theone or more gRNAs hybridize to the target site to guide the Cas nucleaseto cut the target site, thereby killing the first bacteria, wherein thesubject comprises second bacteria of one or more strains or species thatare different from that of the first bacteria, wherein the genomes ofthe second bacteria do not comprise the target site, wherein the genomesof the second bacteria are not cut by the Cas nuclease in the subject,whereby second bacteria survive in the presence of the Cas nuclease inthe subject, and wherein the number of the first bacteria is reduced atleast 100-fold by the first 30 minutes after exposing the subject to theone or more gRNAs.
 15. The method of claim 1, wherein the Cas nucleaseis an endogenous Cas nuclease of the first bacteria.
 16. The method ofclaim 1, wherein the Cas nuclease is a Cpf1, a Cas9, or a Cas3.
 17. Themethod of claim 1, wherein the Cas nuclease is a Cas9.
 18. The method ofclaim 1, wherein the Cas nuclease is a Cas3.
 19. The method of claim 1,wherein the first bacteria are Clostridium.
 20. A method of selectivelykilling treating or reducing the risk of a urinary tract infectioncaused by first bacteria in a subject, the method comprising:selectively killing the first bacteria in the subject by cutting atarget site comprised by the genomes of the first bacteria using anRNA-guided Cas nuclease, wherein the method comprises administering tothe subject a nucleic acid sequence comprising or encoding one or moreguide RNAs (gRNAs) at a first time (T1) and at a second time (T2),wherein T2 is at least 3 hours after T1, wherein the one or more gRNAshybridize to the target site to guide the Cas nuclease to cut the targetsite, thereby killing the first bacteria, wherein the subject comprisessecond bacteria of one or more strains or species that are differentfrom that of the first bacteria, wherein the genomes of the secondbacteria do not comprise the target site, wherein the genomes of thesecond bacteria are not cut by the Cas nuclease in the subject, wherebysecond bacteria survive in the presence of the Cas nuclease in thesubject, and wherein the method comprises maintaining reduction in thenumber of the first bacteria by at least 100-fold for at least 60minutes after exposing the subject to the one or more gRNAs.
 21. Themethod of claim 1, wherein the second bacteria are of one or morespecies that are different from that of the first bacteria.
 22. Themethod of claim 20, wherein the second bacteria are of one or morespecies that are different from that of the first bacteria.
 23. Themethod of claim 20, wherein the nucleic acid sequence is or is comprisedby a vector selected from the group consisting of a phage, phagemid,viriophage, virus, plasmid, and a transposon.
 24. The method of claim20, wherein the nucleic acid sequence is or is comprised by aconjugative plasmid.
 25. The method of claim 20, wherein the Casnuclease is a Cas9.
 26. The method of claim 20, wherein the Cas nucleaseis a Cas3.
 27. The method of claim 1, wherein the subject has beenadministered an immunosuppressant medication.
 28. The method of claim20, wherein the subject has been administered an immunosuppressantmedication.